Plasticity Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
Received: August 22, 2014 Accepted after revision: January 26, 2015 Published online: March 17, 2015
Nitric Oxide Pathway and Proliferation of Neural Progenitors in the Neonatal Rat An Phan Duy a–c Hoa Pham a–c Julien Pansiot a–c Pierre Gressens a–c Christiane Charriaut-Marlangue a–c Olivier Baud a–d a
INSERM, UMR1141, b Faculté de Médecine, Université Paris Diderot, c PremUP Foundation, and d Assistance Publique – Hôpitaux de Paris, Université Paris Diderot, Neonatal Intensive Care Unit, Hôpital Robert Debré, Paris, France
Abstract Several lines of evidence demonstrate that inhaled nitric oxide (iNO) not only acts locally on the pulmonary vasculature but also has remote effects on the mature and developing brain under basal or pathological conditions by modulating cerebral blood flow and microvascularization, white matter maturation, inflammation, and subsequent brain repair. Previously, consistent studies demonstrated that increased levels of guanosine 3′,5′ cyclic monophosphate (cGMP), the main effector of biological effect induced by nitric oxide (NO), significantly augment proliferation and neuronal differentiation of adult neural progenitor cells (NPCs). In the present study, we ask the question whether iNO could promote the proliferation of NPCs in the uninjured developing brain. We first reported that iNO exposure at a concentration of 20 ppm during the first 7 days of life was associated with a significant but transient elevation of brain cGMP concentration 2 h after the onset of iNO exposure and a subsequent increase in myelin content of the developing white matter at postnatal day (P) 10. Using BrDu labelling and colabelling with specific cell-type markers we found that iNO exposure of rat pups results in an increased NPC proliferation in sev-
© 2015 S. Karger AG, Basel 0378–5866/15/0375–0417$39.50/0 E-Mail [email protected] www.karger.com/dne
eral layers of the subventricular zone (SVZ) at both early (30 h) and late (P7) time points. These proliferating NPCs were found to be sustainably viable and subsequently differentiated into oligodendroglial cells in the developing white matter and cortex. We also found that NG2 immunoreactivity around vessel walls, labeling pericyte cells, was increased in NO-exposed rat pups in the periventricular SVZ. In conclusion, iNO appears to act on oligodendrocyte progenitor cells, leading to increased density of mature oligodendrocytes and myelin content in the immature rat brain. © 2015 S. Karger AG, Basel
Introduction
Brain development is a key element of all maturation processes and is essential to ensure the integration of the child and future adult into society. The neonatal period is crucial for brain development, and the newborn developing brain is particularly vulnerable to injury. Cerebral palsy, the most common cause of severe disability in children, is observed in 1–2 out of every 1,000 newborns [1, 2]. Cerebral palsy results from a number of disparate insults to the developing brain, acting alone or in combination [3]. In recent reports, 5–10% of preterm infants developed cerebral palsy and 25–50% had cognitive impairment and behavioral disorders [4]. Strategies to prevent Olivier Baud, MD, PhD INSERM U1141 Hôpital Robert Debré 48 blvd Sérurier, FR–75019 Paris (France) E-Mail olivier.baud @ rdb.aphp.fr
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Key Words Nitric oxide · Neural progenitors · Developing brain · Oligodendrocyte · Myelination
Materials and Methods Ethics Statement All experiments complied with the ethical guidelines of the Robert Debré Hospital Research Council Review Board (A75-1901), which approved this study, and followed guidelines of INSERM and ARRIVE (http://www.nc3rs.org/ARRIVE).
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Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
Experimental Protocol and Gas Exposure Pregnant Sprague-Dawley rats (Charles River Laboratories, France) were placed in normoxic gas chambers (Biospherix, Redfield, N.Y., USA). Oxygen concentration was monitored using a Proox device (Biospherix). To investigate the impact of exogenous inhaled NO on the developing brain, low-concentration iNO (20 ppm) was introduced into the chambers 12–24 h prior to delivery and up to postnatal day (P) 7 and monitored using an iNOvent system (INOTherapeutics, Clinton, N.J., USA). The NO2 concentration was kept under normal concentration of 1 ppm. In the control group, the animals were kept in room air after delivery. The animals were housed under controlled temperature (22 ± 1 ° C) and light conditions (12-hour day/night cycle) with food and water ad libitum.
cGMP Measurement Competitive enzyme immunoassay (Cayman Chemical Company, Ann Arbor, Mich., USA) was used to quantify cGMP in the forebrain, according to the manufacturer’s instructions. Whole brains were harvested at P1 (2 h after the onset of iNO exposure), P3 and P7 and immediately frozen at –80 ° C until measurements were taken.
BrDu Injection and Cell Proliferation Assay Cell proliferation was investigated by injecting BrDu (50 mg/ kg) to rat pups at 26 and 28 h after birth (occurring midnight before). Pups were sacrificed 2 h following the second injection of BrDu to analyze cell proliferation and at P7 to explore cell fate and differentiation of labeled cells in the SVZ, white matter and cortex. Tissue Preparation Both male and female pups were sacrificed 30 h after birth (2 h after the end of BrDu injections) and at p7 (n = 6–9/group for each histological protocol). Anesthesia was with inhaled isoflurane (Abbott France, Rungis, France). Each anesthetized animal received a transcardial infusion of 4% paraformaldehyde in phosphate buffer (0.12 M, pH = 7.4). The brains were harvested, post-fixed in the same fixative for 3 h, equilibrated with 10% sucrose in phosphate buffer for 2–4 days, frozen in liquid-nitrogen-cooled isopentane, and stored at –80 ° C as previously reported [8]. Serial coronal sections 10 μm in thickness were cut on a sagittal plane at 30 h of postnatal age and on a coronal plane at P7. These two experimental procedures were done for a better analysis of the SVZ according to brain size.
Immunohistochemistry Primary antibodies used in this study are listed in online supplementary table 1 (for all online suppl. material, see www.karger. com/doi/10.1159/000375488). All quantification of immunoreactive cells was carried out by investigators blinded to the experimental groups. For brain immunohistochemistry at P7, coronal sections (+1.44 to –0.48 mm from bregma) were selected and processed as previously described [11]. In each experimental group, we studied 7–8 pups in three separate experiments. Immunolabeling was visualized using the streptavidin-biotin-peroxidase method. For BrDu-immunostaining, sections were treated as follows before incubation with the primary antibody: trypsin pretreatment (Sigma) 0.2% in Tris buffer (pH 7.5) and 0.1% calcium chloride for
Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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or attenuate perinatal brain damage are chiefly relevant in the context of hypoxic-ischemic encephalopathy in term or near-term neonates but not in preterm infants. Indeed, to date, most drugs expected to prevent brain damage have been discarded in the clinical setting, especially in the most immature infants [5]. Nitric oxide (NO) is a small water-soluble molecule that can easily diffuse through tissues. Endogenously released NO is also widely recognized as playing a key role in multiple organ systems in vertebrates, including respiratory and central nervous systems during critical phases of development [6, 7]. Data on the neurological follow-up of premature infants exposed to inhaled nitric oxide (iNO) remain sparse and difficult to interpret. However, several lines of evidence demonstrate that low doses of iNO not only act locally on the pulmonary vasculature but also have remote effects on the developing brain under basal or pathological conditions by modulating white matter maturation, inflammation and subsequent brain repair [8]. Indeed, we recently reported that the NO pathway is able to promote oligodendroglial maturation, myelination and neuroprotection in preclinical models of neonatal stroke and white matter damage induced by postnatal hyperoxia or excitotoxic insult [9–12]. Several other reports have demonstrated that iNO is associated with significant neuroprotection in preclinical models of adult brain damage [13–15]. On the other hand, mechanistic aspects of iNO-induced neuroprotection remain unclear. A recent report has shown that EPO, a candidate molecule for neuroprotection in stroke, was associated with enhancement in the proliferation and migration to the site of injury of subventricular zone (SVZ) neural progenitor cells (NPCs) [16]. This study suggests that modulating the cell fate of endogenous NPCs in the developing brain could be an interesting strategy to promote repair. In addition, increasing evidence supports the relationship between the level of guanosine 3′,5′ cyclic monophosphate (cGMP), the main effector of NO biological effects, and the ability of the SVZ to produce NPCs [17]. Here, we ask the question whether iNO could promote the proliferation of NPCs within the SVZ in the uninjured developing brain.
Fig. 1. Impact of iNO on myelination and
Optical Density of Myelinated Fibers and NG2+ Pericytes The optical density of MBP-stained fibers and NG2+ pericytes was measured using a computerized image-analysis system (ImageJ 1.41o; NIH, USA; http://rsb.info.nih.gov/ij/) that reads optical density as gray levels; 4 sections per brain were examined for each animal at P7. Nonspecific background densities were measured at each brain level in a region devoid of MBP or NG2 immunolabeling and were subtracted from values of the region of interest as previously reported [18]. Quantitative Measurements of BrDu and Double-Positive Cells All quantitative measurements were done by observers who were blinded to the experimental group of the animal. After delineating regions of interests within the SVZ areas, white matter and cortex, the density of either BrDu+ cells or BrDu/cell-specific marker double-positive cells was calculated and reported to the anatomic area previously delineated for each section. At least 6 sections per brain were examined. Statistical Analysis Values are expressed as means ± SEM. Statistical comparisons between normoxic control and exposed groups were performed using one-way ANOVA followed by Dunnett’s multiple comparison tests. Pairwise comparisons between groups with or without
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iNO exposure were done using the Mann-Whitney U test. Statistical tests were run on Prism version 5.01 (GraphPad Software, San Diego, Calif., USA) and StatView version 5.0 for Mac OS (Abacus Concepts, Berkeley, Calif., USA).
Results
iNO Increases Myelin Fiber Density within the Developing White Matter and Cortex in the Neonatal Rat As previously reported by Olivier et al. [9], iNO exposure (20 ppm) during the first week of life was associated with a significant increase in myelin fiber density at P7 in several white matter areas, including the lateral corpus callosum in the normal neonatal rat (fig. 1a–c). This effect was found more pronounced in, but not restricted to, periventricular white matter (p < 0.001) and was also observed in the myelinated fibers detected in the cortex (p < 0.05). Exposure to NO induced a significant and rapid (within 2 h) elevation of brain cGMP concentration in P1 animals, suggesting that exogenous NO was able to reach the developing brain and stimulate guanylate cyclase activity (fig. 1d). This effect was only transient and no longer observed at P3 and P7 (brain concentrations have been found below the detection limit at these time points Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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NO and Neural Progenitors
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15 min at 37 ° C, DNA denaturation step in 2N HCl for 30 min at room temperature, and two 30-min washes in 0.1 M lysine (pH 6). Double-labeling was performed with secondary antibodies coupled to the green fluorescent marker Fluoroprobe S488 (Interchim, Montluçon, France) or the red fluorescent marker cyanine 3 (Jackson ImmunoResearch laboratories, West Grove, Pa., USA).
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cGMP level in the neonatal rat brain. OD = Optical density. a, b Photomicrographs showing MBP+ fibers in 2 regions of interest (lateral white matter and cortex) in P7 rat pups exposed to air and to iNO 20 ppm. Scale bar = 200 μm. c Quantitative analysis of the optical density of MBP+ fibers in the lateral white matter and cortex in P7 rat pups with or without exposure to iNO (20 ppm) compared to controls (air). d Quantitative analysis of cGMP brain concentrations in P1, P3 and P7 rat pups subjected to iNO exposure. * p < 0.05, *** p < 0.001, using Mann-Whitney test for comparisons between air- and iNO-exposed groups.
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Fig. 2. Impact of iNO on early proliferative activity of NPCs within several areas of the SVZ. LV = Lateral ventricle. a Photomicrograph showing where SVZ regions of interest are located in a parasagittal section of rat brain 30 h after birth. We designed 6 areas of interest, including septal SVZ (area 1), neocortical SVZ (area 2), white matter (area 3), cortical plate (area 4), septal neuroepithelium (area 5), and rhinencephalic SVZ (area 6). b Quantitative anal-
ysis of BrDu+ cell density in various subventricular areas of interest. c, d Photomicrographs showing BrDu immunoreactivity in H30 rat pups with or without exposure to iNO. White lines delineated the anatomic surface of the SVZ (area 5) for density calculation. Scale bar = 50 μm. *** p < 0.001, for comparisons using oneway ANOVA test followed by Dunnett’s multiple comparison tests.
with and without iNO). These data were consistent with the changes of NO concentration detected by the voltammetric method in the developing brain following iNO, which we have previously reported [11].
1), neocortical SVZ (area 2), white matter (area 3), cortical plate (area 4), septal neuroepithelium (area 5), and rhinencephalic SVZ (area 6) (fig. 2a). As expected, the density of NPCs was found higher in the SVZ zones in air-exposed animals. iNO exposure initiated just before birth was associated with a significant increase in proliferating NPC density in the septal SVZ and neuroepithelium (fig. 2b–d).
iNO Induces Early Proliferative Effect on NPCs in SVZ Because the exposure to iNO was found to be associated with a proliferative effect on immature oligodendrocytes [9], we asked the question whether iNO could have a significant impact on NPCs proliferation within several layers of the SVZ, white matter and cortex very early after the onset of iNO exposure. To address this question, we have chosen to examine the P1 brain on sagittal sections, showing larger areas of the SVZ. We designed 6 areas of interest, including septal SVZ (area 420
Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
iNO Induces Protracted Proliferative Effect on NPCs in SVZ and Developing White Matter To determine whether the effect of iNO on NPC proliferation is a transient or a protracted phenomenon, the density of BrDu+ cells was measured at P7. At that developmental stage, we explored the developing brain on the Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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Fig. 3. Impact of iNO on proliferative activ-
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iNO-Induced NPC Fate and Differentiation To examine the cell fate of NPCs produced in response to iNO exposure, we first calculated the proportion of BrDu+ cells in areas described above that colabeled with cleaved caspase-3, a marker of dying cells (fig. 4). We found that caspase-3/BrDu double-positive cell density relative to total BrDu+ cell density was found unchanged or reduced in most of areas studied (fig. 4a, b). Therefore, newly formed NPCs generated after birth in response to iNO exposure were not more likely to die than those in control animals at P7. Next, we investigated cell differentiation of these NPCs to determine whether iNO exposure resulted in a favorable effect on the gen-
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eration of oligodendrocytes and subsequent myelination. Colabeling using markers for neuronal (NeuN) and astrocyte (PS100) cells were used in the white matter and cortical plate. Olig2, as a marker for oligodendrocyte precursors, was used in SVZ areas, white matter and cortex. Colabeled NeuN+/BrDu+ cells were not detected in the cortex in both iNO-exposed and room air-exposed animals at P7 (fig. 5). In contrast, PS100+ cells were found to have an increased density in iNO-treated animals compared to controls in the white matter and cortical plate (fig. 6a). However, the proportion of double-positive cells relative to all BrDu+ cells was found similar in the two groups (fig. 6b–d). There was an increased density of colabeled Olig2+/BrDu+ oligodendrocytes in the cortex (p < 0.001), striatum (p < 0.001) and white matter (p < 0.05) in response to iNO exposure in P7 rat pups (fig. 7). Finally, to further confirm that oligodendroglial progenitor density was increased in iNO-exposed animals at P7, we performed double immunofluorescent staining using the NG2 marker together with BrDu in the white matter and cortical plate. We found that NG2/ BrDu double-positive cells were in higher proportion in the treated animals relative to controls in these areas (fig. 8a). In addition, we found that NG2 immunoreactivity around vessel walls, labeling pericyte cells, was increased in NO-exposed rat pups in the periventricular SVZ (fig. 8b–d). Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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a
coronal section, delineating the following 6 areas of interest: neocortical SVZ (area 1), striatal neuroepithelium and SVZ (area 2), striatal SVZ and lateral migratory stream (area 3), white matter (area 4), cortical plate, infragranular part (area 5) and supragranular part (area 5bis), and striatum (area 6; fig. 3a). Adjusted to the surface of interest, NPC density was found significantly increased in animals subjected to iNO from birth to P7 relative to air-exposed animals (fig. 3b). In addition, a higher density of labeled NPCs was detected in white matter but not in the cortical plate (fig. 3c). A small but significant increase in BrDu+ NPCs was also noted in the striatum in iNO-exposed P7 rat pups (fig. 3c).
NO and Neural Progenitors
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ity of NPCs within SVZs and cortical areas in P7 rat pups. a Photomicrograph showing where SVZ regions of interest are located in a coronal section of P7 rat brain. We designed 7 areas of interest, including neocortical SVZ (area 1), striatal neuroepithelium and SVZ (area 2), striatal SVZ and lateral migratory stream (area 3), white matter (area 4), cortical plate, infragranular part (area 5) and supragranular part (area 5bis), and striatum (area 6). b Quantitative analysis of BrDu+ cell density in various subventricular areas of interest, with or without exposure to iNO. c Enlarged graph showing quantitative analysis of BrDu+ cell density in the white matter, cortical plate and basal ganglia, with or without exposure to iNO. * p < 0.05, *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
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Fig. 4. Cell death of BrdU-positive NPCs in P7 rat pups. a, b Quantitative analysis of BrdU/cleaved caspase-3 double-positive cell density and its ratio to total BrdU+ cells ratio in various subventricular areas of interest, with or without exposure to iNO. c, d Photomicrograph showing an example of double immunos-
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taining using BrdU (green) and cleaved caspase-3 (red), with BrdU cells (arrows), caspase-3+ cells (asterisks) and double-positive cells (arrowheads). Scale bar = 30 μm. ** p < 0.01, *** p < 0.001, for comparisons using one-way ANOVA test followed by the Dunnett’s multiple comparison tests.
Fig. 5. BrDu+ NPCs are not neuronal in the cortical plate. Photomicrograph at 2 magnifications showing an example of double immunostaining using BrDu (green) and NeuN (red), with BrDu+ cells (arrows) and NeuN+ cells (asterisks). None of the BrDu+ cells were found to be NeuN+. Scale bar = 20 μm.
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Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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ble-positive cell density and its ratio to total BrDu+ cells in the white matter and cortical plate, with or without exposure to iNO. c, d Photomicrographs showing double immunostaining using
Altogether, these data suggest that iNO is able to promote proliferation of NPCs that are sustainably viable and that subsequently differentiate into oligodendroglial cells in the developing white matter and cortex.
Discussion
This present study demonstrates that iNO exposure of rat pups at a concentration of 20 ppm, relevant in clinical practice, results in an increased NPC proliferation at both early (30 h) and late (P7) time points. These cells migrate into the developing white matter and cortical plate and preferentially differentiate into oligodendroglial cells. NO and Neural Progenitors
BrDu (green) and PS100 (red), with BrDu+ cells (arrows), PS100+ cells (asterisks) and double-positive cells (arrowheads), in the white matter (c) and cortical plate (d). Scale bar = 30 μm. *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
Inhaled NO is increasingly considered as a promising molecule acting as a neuroprotectant in adults and juvenile rodents [8, 19]. We previously reported a significant effect of both exogenous and endogenous NO on myelination in immature normal rodents [9] and, in particular for iNO, a direct impact on proliferating oligodendroglial cells and their maturation into myelinating cells. In addition, we and others consistently reported that iNO is able to promote neuroprotective properties in several models of brain injury both in the adult and neonatal rat [8]. Indeed, several lines of evidence demonstrate that low doses of iNO modulate cerebral blood flow and microvascularization, white matter maturation, inflammation, and subsequent brain repair. Inhaled NO, a drug approved in 2001 for therapeutic use in neonates with hyDev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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Fig. 6. BrDu+ NPCs and the glial marker PS100 in white matter and cortical plate. a, b Quantitative analysis of BrDu/PS100 dou-
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Fig. 7. BrDu+ NPCs and the oligodendroglial marker Olig2 in the
SVZ, white matter and cortical plate. v: Vessels (proliferating endothelial cells). a, b Quantitative analysis of BrDu/Olig2 doublepositive cell density and its ratio to total BrDu+ cells in the white matter and cortical plate, with or without exposure to iNO. c, d Photomicrographs showing double immunostaining using
poxic respiratory failure by the European Medicine Evaluation Agency and the European Commission, could therefore be effective in preventing brain damage and/or enhancing repair. However, mechanistic aspects, biomarkers and preclinical setup must be further studied before clinical translation. Here, we investigated a significant impact of iNO on SVZ NPC-derived neurogenic activity in the developing brain. The present study explored the first step of a new potential mechanism leading to neuroprotection following iNO exposure. In the SVZ of the lateral ventricle, a generation of NPCs that differentiate into neurons, astrocytes and oligodendrocytes occurs throughout life. 424
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Area 5
BrDu (green) and Olig2 (red), with BrDu+ cells (arrows), olig2positive cells (asterisks) and double-positive cells (arrowheads), in the white matter (c) and cortical plate (d). Scale bar = 30 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
A key role for NO has been reported in the nervous system as a coordinator of proliferation and patterning during neural development and adult neurogenesis [20]. NO has been reported as a negative regulator of cell proliferation in the mammalian brain [21–25]. However, most of these studies investigated the adult mouse or focused on cerebellar precursor cells. Negative effects have been attributed to the neuronal isoform of NO synthase in nitrergic neurons [22]. In contrast, after brain damage, NO overproduction in different neural and nonneural cell types promotes neurogenesis. In addition, some reports suggest that a decrease in basal levels of cGMP and an increase in phosphodiesterase Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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and cortical plate and pericyte density in the SVZ. OD = Optical density; LV = lateral ventricle. a Quantitative analysis of BrDu/ NG2 double-positive cells/BrDu+ cell ratio in the white matter and cortical plate, with or without exposure to iNO. * p < 0.05, ** p
Received: August 22, 2014 Accepted after revision: January 26, 2015 Published online: March 17, 2015
Nitric Oxide Pathway and Proliferation of Neural Progenitors in the Neonatal Rat An Phan Duy a–c Hoa Pham a–c Julien Pansiot a–c Pierre Gressens a–c Christiane Charriaut-Marlangue a–c Olivier Baud a–d a
INSERM, UMR1141, b Faculté de Médecine, Université Paris Diderot, c PremUP Foundation, and d Assistance Publique – Hôpitaux de Paris, Université Paris Diderot, Neonatal Intensive Care Unit, Hôpital Robert Debré, Paris, France
Abstract Several lines of evidence demonstrate that inhaled nitric oxide (iNO) not only acts locally on the pulmonary vasculature but also has remote effects on the mature and developing brain under basal or pathological conditions by modulating cerebral blood flow and microvascularization, white matter maturation, inflammation, and subsequent brain repair. Previously, consistent studies demonstrated that increased levels of guanosine 3′,5′ cyclic monophosphate (cGMP), the main effector of biological effect induced by nitric oxide (NO), significantly augment proliferation and neuronal differentiation of adult neural progenitor cells (NPCs). In the present study, we ask the question whether iNO could promote the proliferation of NPCs in the uninjured developing brain. We first reported that iNO exposure at a concentration of 20 ppm during the first 7 days of life was associated with a significant but transient elevation of brain cGMP concentration 2 h after the onset of iNO exposure and a subsequent increase in myelin content of the developing white matter at postnatal day (P) 10. Using BrDu labelling and colabelling with specific cell-type markers we found that iNO exposure of rat pups results in an increased NPC proliferation in sev-
© 2015 S. Karger AG, Basel 0378–5866/15/0375–0417$39.50/0 E-Mail [email protected] www.karger.com/dne
eral layers of the subventricular zone (SVZ) at both early (30 h) and late (P7) time points. These proliferating NPCs were found to be sustainably viable and subsequently differentiated into oligodendroglial cells in the developing white matter and cortex. We also found that NG2 immunoreactivity around vessel walls, labeling pericyte cells, was increased in NO-exposed rat pups in the periventricular SVZ. In conclusion, iNO appears to act on oligodendrocyte progenitor cells, leading to increased density of mature oligodendrocytes and myelin content in the immature rat brain. © 2015 S. Karger AG, Basel
Introduction
Brain development is a key element of all maturation processes and is essential to ensure the integration of the child and future adult into society. The neonatal period is crucial for brain development, and the newborn developing brain is particularly vulnerable to injury. Cerebral palsy, the most common cause of severe disability in children, is observed in 1–2 out of every 1,000 newborns [1, 2]. Cerebral palsy results from a number of disparate insults to the developing brain, acting alone or in combination [3]. In recent reports, 5–10% of preterm infants developed cerebral palsy and 25–50% had cognitive impairment and behavioral disorders [4]. Strategies to prevent Olivier Baud, MD, PhD INSERM U1141 Hôpital Robert Debré 48 blvd Sérurier, FR–75019 Paris (France) E-Mail olivier.baud @ rdb.aphp.fr
Downloaded by: Dahlgree Med. Library, Goergetown Univ. 198.143.37.65 - 8/25/2015 8:18:52 PM
Key Words Nitric oxide · Neural progenitors · Developing brain · Oligodendrocyte · Myelination
Materials and Methods Ethics Statement All experiments complied with the ethical guidelines of the Robert Debré Hospital Research Council Review Board (A75-1901), which approved this study, and followed guidelines of INSERM and ARRIVE (http://www.nc3rs.org/ARRIVE).
418
Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
Experimental Protocol and Gas Exposure Pregnant Sprague-Dawley rats (Charles River Laboratories, France) were placed in normoxic gas chambers (Biospherix, Redfield, N.Y., USA). Oxygen concentration was monitored using a Proox device (Biospherix). To investigate the impact of exogenous inhaled NO on the developing brain, low-concentration iNO (20 ppm) was introduced into the chambers 12–24 h prior to delivery and up to postnatal day (P) 7 and monitored using an iNOvent system (INOTherapeutics, Clinton, N.J., USA). The NO2 concentration was kept under normal concentration of 1 ppm. In the control group, the animals were kept in room air after delivery. The animals were housed under controlled temperature (22 ± 1 ° C) and light conditions (12-hour day/night cycle) with food and water ad libitum.
cGMP Measurement Competitive enzyme immunoassay (Cayman Chemical Company, Ann Arbor, Mich., USA) was used to quantify cGMP in the forebrain, according to the manufacturer’s instructions. Whole brains were harvested at P1 (2 h after the onset of iNO exposure), P3 and P7 and immediately frozen at –80 ° C until measurements were taken.
BrDu Injection and Cell Proliferation Assay Cell proliferation was investigated by injecting BrDu (50 mg/ kg) to rat pups at 26 and 28 h after birth (occurring midnight before). Pups were sacrificed 2 h following the second injection of BrDu to analyze cell proliferation and at P7 to explore cell fate and differentiation of labeled cells in the SVZ, white matter and cortex. Tissue Preparation Both male and female pups were sacrificed 30 h after birth (2 h after the end of BrDu injections) and at p7 (n = 6–9/group for each histological protocol). Anesthesia was with inhaled isoflurane (Abbott France, Rungis, France). Each anesthetized animal received a transcardial infusion of 4% paraformaldehyde in phosphate buffer (0.12 M, pH = 7.4). The brains were harvested, post-fixed in the same fixative for 3 h, equilibrated with 10% sucrose in phosphate buffer for 2–4 days, frozen in liquid-nitrogen-cooled isopentane, and stored at –80 ° C as previously reported [8]. Serial coronal sections 10 μm in thickness were cut on a sagittal plane at 30 h of postnatal age and on a coronal plane at P7. These two experimental procedures were done for a better analysis of the SVZ according to brain size.
Immunohistochemistry Primary antibodies used in this study are listed in online supplementary table 1 (for all online suppl. material, see www.karger. com/doi/10.1159/000375488). All quantification of immunoreactive cells was carried out by investigators blinded to the experimental groups. For brain immunohistochemistry at P7, coronal sections (+1.44 to –0.48 mm from bregma) were selected and processed as previously described [11]. In each experimental group, we studied 7–8 pups in three separate experiments. Immunolabeling was visualized using the streptavidin-biotin-peroxidase method. For BrDu-immunostaining, sections were treated as follows before incubation with the primary antibody: trypsin pretreatment (Sigma) 0.2% in Tris buffer (pH 7.5) and 0.1% calcium chloride for
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or attenuate perinatal brain damage are chiefly relevant in the context of hypoxic-ischemic encephalopathy in term or near-term neonates but not in preterm infants. Indeed, to date, most drugs expected to prevent brain damage have been discarded in the clinical setting, especially in the most immature infants [5]. Nitric oxide (NO) is a small water-soluble molecule that can easily diffuse through tissues. Endogenously released NO is also widely recognized as playing a key role in multiple organ systems in vertebrates, including respiratory and central nervous systems during critical phases of development [6, 7]. Data on the neurological follow-up of premature infants exposed to inhaled nitric oxide (iNO) remain sparse and difficult to interpret. However, several lines of evidence demonstrate that low doses of iNO not only act locally on the pulmonary vasculature but also have remote effects on the developing brain under basal or pathological conditions by modulating white matter maturation, inflammation and subsequent brain repair [8]. Indeed, we recently reported that the NO pathway is able to promote oligodendroglial maturation, myelination and neuroprotection in preclinical models of neonatal stroke and white matter damage induced by postnatal hyperoxia or excitotoxic insult [9–12]. Several other reports have demonstrated that iNO is associated with significant neuroprotection in preclinical models of adult brain damage [13–15]. On the other hand, mechanistic aspects of iNO-induced neuroprotection remain unclear. A recent report has shown that EPO, a candidate molecule for neuroprotection in stroke, was associated with enhancement in the proliferation and migration to the site of injury of subventricular zone (SVZ) neural progenitor cells (NPCs) [16]. This study suggests that modulating the cell fate of endogenous NPCs in the developing brain could be an interesting strategy to promote repair. In addition, increasing evidence supports the relationship between the level of guanosine 3′,5′ cyclic monophosphate (cGMP), the main effector of NO biological effects, and the ability of the SVZ to produce NPCs [17]. Here, we ask the question whether iNO could promote the proliferation of NPCs within the SVZ in the uninjured developing brain.
Fig. 1. Impact of iNO on myelination and
Optical Density of Myelinated Fibers and NG2+ Pericytes The optical density of MBP-stained fibers and NG2+ pericytes was measured using a computerized image-analysis system (ImageJ 1.41o; NIH, USA; http://rsb.info.nih.gov/ij/) that reads optical density as gray levels; 4 sections per brain were examined for each animal at P7. Nonspecific background densities were measured at each brain level in a region devoid of MBP or NG2 immunolabeling and were subtracted from values of the region of interest as previously reported [18]. Quantitative Measurements of BrDu and Double-Positive Cells All quantitative measurements were done by observers who were blinded to the experimental group of the animal. After delineating regions of interests within the SVZ areas, white matter and cortex, the density of either BrDu+ cells or BrDu/cell-specific marker double-positive cells was calculated and reported to the anatomic area previously delineated for each section. At least 6 sections per brain were examined. Statistical Analysis Values are expressed as means ± SEM. Statistical comparisons between normoxic control and exposed groups were performed using one-way ANOVA followed by Dunnett’s multiple comparison tests. Pairwise comparisons between groups with or without
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iNO exposure were done using the Mann-Whitney U test. Statistical tests were run on Prism version 5.01 (GraphPad Software, San Diego, Calif., USA) and StatView version 5.0 for Mac OS (Abacus Concepts, Berkeley, Calif., USA).
Results
iNO Increases Myelin Fiber Density within the Developing White Matter and Cortex in the Neonatal Rat As previously reported by Olivier et al. [9], iNO exposure (20 ppm) during the first week of life was associated with a significant increase in myelin fiber density at P7 in several white matter areas, including the lateral corpus callosum in the normal neonatal rat (fig. 1a–c). This effect was found more pronounced in, but not restricted to, periventricular white matter (p < 0.001) and was also observed in the myelinated fibers detected in the cortex (p < 0.05). Exposure to NO induced a significant and rapid (within 2 h) elevation of brain cGMP concentration in P1 animals, suggesting that exogenous NO was able to reach the developing brain and stimulate guanylate cyclase activity (fig. 1d). This effect was only transient and no longer observed at P3 and P7 (brain concentrations have been found below the detection limit at these time points Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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NO and Neural Progenitors
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15 min at 37 ° C, DNA denaturation step in 2N HCl for 30 min at room temperature, and two 30-min washes in 0.1 M lysine (pH 6). Double-labeling was performed with secondary antibodies coupled to the green fluorescent marker Fluoroprobe S488 (Interchim, Montluçon, France) or the red fluorescent marker cyanine 3 (Jackson ImmunoResearch laboratories, West Grove, Pa., USA).
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cGMP (pmol/ml)
cGMP level in the neonatal rat brain. OD = Optical density. a, b Photomicrographs showing MBP+ fibers in 2 regions of interest (lateral white matter and cortex) in P7 rat pups exposed to air and to iNO 20 ppm. Scale bar = 200 μm. c Quantitative analysis of the optical density of MBP+ fibers in the lateral white matter and cortex in P7 rat pups with or without exposure to iNO (20 ppm) compared to controls (air). d Quantitative analysis of cGMP brain concentrations in P1, P3 and P7 rat pups subjected to iNO exposure. * p < 0.05, *** p < 0.001, using Mann-Whitney test for comparisons between air- and iNO-exposed groups.
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Fig. 2. Impact of iNO on early proliferative activity of NPCs within several areas of the SVZ. LV = Lateral ventricle. a Photomicrograph showing where SVZ regions of interest are located in a parasagittal section of rat brain 30 h after birth. We designed 6 areas of interest, including septal SVZ (area 1), neocortical SVZ (area 2), white matter (area 3), cortical plate (area 4), septal neuroepithelium (area 5), and rhinencephalic SVZ (area 6). b Quantitative anal-
ysis of BrDu+ cell density in various subventricular areas of interest. c, d Photomicrographs showing BrDu immunoreactivity in H30 rat pups with or without exposure to iNO. White lines delineated the anatomic surface of the SVZ (area 5) for density calculation. Scale bar = 50 μm. *** p < 0.001, for comparisons using oneway ANOVA test followed by Dunnett’s multiple comparison tests.
with and without iNO). These data were consistent with the changes of NO concentration detected by the voltammetric method in the developing brain following iNO, which we have previously reported [11].
1), neocortical SVZ (area 2), white matter (area 3), cortical plate (area 4), septal neuroepithelium (area 5), and rhinencephalic SVZ (area 6) (fig. 2a). As expected, the density of NPCs was found higher in the SVZ zones in air-exposed animals. iNO exposure initiated just before birth was associated with a significant increase in proliferating NPC density in the septal SVZ and neuroepithelium (fig. 2b–d).
iNO Induces Early Proliferative Effect on NPCs in SVZ Because the exposure to iNO was found to be associated with a proliferative effect on immature oligodendrocytes [9], we asked the question whether iNO could have a significant impact on NPCs proliferation within several layers of the SVZ, white matter and cortex very early after the onset of iNO exposure. To address this question, we have chosen to examine the P1 brain on sagittal sections, showing larger areas of the SVZ. We designed 6 areas of interest, including septal SVZ (area 420
Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
iNO Induces Protracted Proliferative Effect on NPCs in SVZ and Developing White Matter To determine whether the effect of iNO on NPC proliferation is a transient or a protracted phenomenon, the density of BrDu+ cells was measured at P7. At that developmental stage, we explored the developing brain on the Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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Fig. 3. Impact of iNO on proliferative activ-
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iNO-Induced NPC Fate and Differentiation To examine the cell fate of NPCs produced in response to iNO exposure, we first calculated the proportion of BrDu+ cells in areas described above that colabeled with cleaved caspase-3, a marker of dying cells (fig. 4). We found that caspase-3/BrDu double-positive cell density relative to total BrDu+ cell density was found unchanged or reduced in most of areas studied (fig. 4a, b). Therefore, newly formed NPCs generated after birth in response to iNO exposure were not more likely to die than those in control animals at P7. Next, we investigated cell differentiation of these NPCs to determine whether iNO exposure resulted in a favorable effect on the gen-
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eration of oligodendrocytes and subsequent myelination. Colabeling using markers for neuronal (NeuN) and astrocyte (PS100) cells were used in the white matter and cortical plate. Olig2, as a marker for oligodendrocyte precursors, was used in SVZ areas, white matter and cortex. Colabeled NeuN+/BrDu+ cells were not detected in the cortex in both iNO-exposed and room air-exposed animals at P7 (fig. 5). In contrast, PS100+ cells were found to have an increased density in iNO-treated animals compared to controls in the white matter and cortical plate (fig. 6a). However, the proportion of double-positive cells relative to all BrDu+ cells was found similar in the two groups (fig. 6b–d). There was an increased density of colabeled Olig2+/BrDu+ oligodendrocytes in the cortex (p < 0.001), striatum (p < 0.001) and white matter (p < 0.05) in response to iNO exposure in P7 rat pups (fig. 7). Finally, to further confirm that oligodendroglial progenitor density was increased in iNO-exposed animals at P7, we performed double immunofluorescent staining using the NG2 marker together with BrDu in the white matter and cortical plate. We found that NG2/ BrDu double-positive cells were in higher proportion in the treated animals relative to controls in these areas (fig. 8a). In addition, we found that NG2 immunoreactivity around vessel walls, labeling pericyte cells, was increased in NO-exposed rat pups in the periventricular SVZ (fig. 8b–d). Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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a
coronal section, delineating the following 6 areas of interest: neocortical SVZ (area 1), striatal neuroepithelium and SVZ (area 2), striatal SVZ and lateral migratory stream (area 3), white matter (area 4), cortical plate, infragranular part (area 5) and supragranular part (area 5bis), and striatum (area 6; fig. 3a). Adjusted to the surface of interest, NPC density was found significantly increased in animals subjected to iNO from birth to P7 relative to air-exposed animals (fig. 3b). In addition, a higher density of labeled NPCs was detected in white matter but not in the cortical plate (fig. 3c). A small but significant increase in BrDu+ NPCs was also noted in the striatum in iNO-exposed P7 rat pups (fig. 3c).
NO and Neural Progenitors
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ity of NPCs within SVZs and cortical areas in P7 rat pups. a Photomicrograph showing where SVZ regions of interest are located in a coronal section of P7 rat brain. We designed 7 areas of interest, including neocortical SVZ (area 1), striatal neuroepithelium and SVZ (area 2), striatal SVZ and lateral migratory stream (area 3), white matter (area 4), cortical plate, infragranular part (area 5) and supragranular part (area 5bis), and striatum (area 6). b Quantitative analysis of BrDu+ cell density in various subventricular areas of interest, with or without exposure to iNO. c Enlarged graph showing quantitative analysis of BrDu+ cell density in the white matter, cortical plate and basal ganglia, with or without exposure to iNO. * p < 0.05, *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
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Fig. 4. Cell death of BrdU-positive NPCs in P7 rat pups. a, b Quantitative analysis of BrdU/cleaved caspase-3 double-positive cell density and its ratio to total BrdU+ cells ratio in various subventricular areas of interest, with or without exposure to iNO. c, d Photomicrograph showing an example of double immunos-
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taining using BrdU (green) and cleaved caspase-3 (red), with BrdU cells (arrows), caspase-3+ cells (asterisks) and double-positive cells (arrowheads). Scale bar = 30 μm. ** p < 0.01, *** p < 0.001, for comparisons using one-way ANOVA test followed by the Dunnett’s multiple comparison tests.
Fig. 5. BrDu+ NPCs are not neuronal in the cortical plate. Photomicrograph at 2 magnifications showing an example of double immunostaining using BrDu (green) and NeuN (red), with BrDu+ cells (arrows) and NeuN+ cells (asterisks). None of the BrDu+ cells were found to be NeuN+. Scale bar = 20 μm.
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ble-positive cell density and its ratio to total BrDu+ cells in the white matter and cortical plate, with or without exposure to iNO. c, d Photomicrographs showing double immunostaining using
Altogether, these data suggest that iNO is able to promote proliferation of NPCs that are sustainably viable and that subsequently differentiate into oligodendroglial cells in the developing white matter and cortex.
Discussion
This present study demonstrates that iNO exposure of rat pups at a concentration of 20 ppm, relevant in clinical practice, results in an increased NPC proliferation at both early (30 h) and late (P7) time points. These cells migrate into the developing white matter and cortical plate and preferentially differentiate into oligodendroglial cells. NO and Neural Progenitors
BrDu (green) and PS100 (red), with BrDu+ cells (arrows), PS100+ cells (asterisks) and double-positive cells (arrowheads), in the white matter (c) and cortical plate (d). Scale bar = 30 μm. *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
Inhaled NO is increasingly considered as a promising molecule acting as a neuroprotectant in adults and juvenile rodents [8, 19]. We previously reported a significant effect of both exogenous and endogenous NO on myelination in immature normal rodents [9] and, in particular for iNO, a direct impact on proliferating oligodendroglial cells and their maturation into myelinating cells. In addition, we and others consistently reported that iNO is able to promote neuroprotective properties in several models of brain injury both in the adult and neonatal rat [8]. Indeed, several lines of evidence demonstrate that low doses of iNO modulate cerebral blood flow and microvascularization, white matter maturation, inflammation, and subsequent brain repair. Inhaled NO, a drug approved in 2001 for therapeutic use in neonates with hyDev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
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Fig. 6. BrDu+ NPCs and the glial marker PS100 in white matter and cortical plate. a, b Quantitative analysis of BrDu/PS100 dou-
BrDu/Olig2 double-positive cells/0.16 mm2
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Fig. 7. BrDu+ NPCs and the oligodendroglial marker Olig2 in the
SVZ, white matter and cortical plate. v: Vessels (proliferating endothelial cells). a, b Quantitative analysis of BrDu/Olig2 doublepositive cell density and its ratio to total BrDu+ cells in the white matter and cortical plate, with or without exposure to iNO. c, d Photomicrographs showing double immunostaining using
poxic respiratory failure by the European Medicine Evaluation Agency and the European Commission, could therefore be effective in preventing brain damage and/or enhancing repair. However, mechanistic aspects, biomarkers and preclinical setup must be further studied before clinical translation. Here, we investigated a significant impact of iNO on SVZ NPC-derived neurogenic activity in the developing brain. The present study explored the first step of a new potential mechanism leading to neuroprotection following iNO exposure. In the SVZ of the lateral ventricle, a generation of NPCs that differentiate into neurons, astrocytes and oligodendrocytes occurs throughout life. 424
Dev Neurosci 2015;37:417–427 DOI: 10.1159/000375488
Area 5
BrDu (green) and Olig2 (red), with BrDu+ cells (arrows), olig2positive cells (asterisks) and double-positive cells (arrowheads), in the white matter (c) and cortical plate (d). Scale bar = 30 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, for comparisons using one-way ANOVA test followed by Dunnett’s multiple comparison tests.
A key role for NO has been reported in the nervous system as a coordinator of proliferation and patterning during neural development and adult neurogenesis [20]. NO has been reported as a negative regulator of cell proliferation in the mammalian brain [21–25]. However, most of these studies investigated the adult mouse or focused on cerebellar precursor cells. Negative effects have been attributed to the neuronal isoform of NO synthase in nitrergic neurons [22]. In contrast, after brain damage, NO overproduction in different neural and nonneural cell types promotes neurogenesis. In addition, some reports suggest that a decrease in basal levels of cGMP and an increase in phosphodiesterase Phan Duy/Pham/Pansiot/Gressens/ Charriaut-Marlangue/Baud
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and cortical plate and pericyte density in the SVZ. OD = Optical density; LV = lateral ventricle. a Quantitative analysis of BrDu/ NG2 double-positive cells/BrDu+ cell ratio in the white matter and cortical plate, with or without exposure to iNO. * p < 0.05, ** p
