Prenatal opioid exposure: The next neonatal neuroinflammatory disease.

The rates of opioid use disorder during pregnancy have more than quadrupled in the last decade, resulting in numerous infants suffering exposure to opioids during the perinatal period, a critical period of central nervous system (CNS) development. Despite increasing use, the characterization and definition of the molecular and cellular mechanisms of the long-term neurodevelopmental impacts of opioid exposure commencing in utero remains incomplete. Thus, in consideration of the looming public health crisis stemming from the multitude of infants with prenatal opioid exposure entering school age, we undertook an investigation of the effects of perinatal methadone exposure in a novel preclinical model. Specifically, we examined the effects of opioids on the developing brain to elucidate mechanisms of putative neural cell injury, to identify diagnostic biomarkers and to guide clinical studies of outcome and follow-up. We hypothesized that methadone would induce a pronounced inflammatory profile in both dams and their pups, and be associated with immune system dysfunction, sustained CNS injury, and altered cognition and executive function into adulthood. This investigation was conducted using a combination of cellular, molecular, biochemical, and clinically translatable biomarker, imaging and cognitive assessment platforms. Data reveal that perinatal methadone exposure increases inflammatory cytokines in the neonatal peripheral circulation, and reprograms and primes the immune system through sustained peripheral immune hyperreactivity. In the brain, perinatal methadone exposure not only increases chemokines and cytokines throughout a crucial developmental period, but also alters microglia morphology consistent with activation, and upregulates TLR4 and MyD88 mRNA. This increase in neuroinflammation coincides with reduced myelin basic protein and altered neurofilament expression, as well as reduced structural coherence and significantly decreased fractional anisotropy on diffusion tensor imaging. In addition to this microstructural brain injury, adult rats exposed to methadone in the perinatal period have significant impairment in associative learning and executive control as assessed using touchscreen technology. Collectively, these data reveal a distinct systemic and neuroinflammatory signature associated with prenatal methadone exposure, suggestive of an altered CNS microenvironment, dysregulated developmental homeostasis, complex concurrent neural injury, and imaging and cognitive findings consistent with clinical literature. Further investigation is required to define appropriate therapies targeted at the neural injury and improve the long-term outcomes for this exceedingly vulnerable patient population.

Chorioamnionitis in Rats Precipitates Extended Postnatal Inflammatory Lymphocyte Hyperreactivity.

Preterm birth is an important cause of perinatal brain injury (PBI). Neurological injury in extremely preterm infants often begins in utero with chorioamnionitis (CHORIO) or inflammation/infection of the placenta and concomitant placental insufficiency. Studies in humans have shown dysregulated inflammatory signaling throughout the placental-fetal brain axis and altered peripheral immune responses in children born preterm with cerebral palsy (CP). We hypothesized that peripheral immune responses would be altered in our well-established rat model of CP. Specifically, we proposed that isolated peripheral blood mononuclear cells (PBMCs) would be hyperresponsive to a second hit of inflammation throughout an extended postnatal time course. Pregnant Sprague-Dawley dams underwent a laparotomy on embryonic day 18 (E18) with occlusion of the uterine arteries (for 60 min) followed by intra-amniotic injection of lipopolysaccharide (LPS, 4 µg/sac) to induce injury in utero. Shams underwent laparotomy only, with equivalent duration of anesthesia. Laparotomies were then closed, and the rat pups were born at E22. PBMCs were isolated from pups on postnatal day 7 (P7) and P21, and subsequently stimulated in vitro with LPS for 3 or 24 h. A secreted inflammatory profile analysis of conditioned media was performed using multiplex electrochemiluminescent immunoassays, and the composition of inflammatory cells was assayed with flow cytometry (FC). Results indicate that CHORIO PBMCs challenged with LPS are hyperreactive and secrete significantly more tumor necrosis factor α (TNFα) and C-X-C chemokine ligand 1 at P7. FC confirmed increased intracellular TNFα in CHORIO pups at P7 following LPS stimulation, in addition to increased numbers of CD11b/c immunopositive myeloid cells. Notably, TNFα secretion was sustained until P21, with increased interleukin 6, concomitant with increased expression of integrin ß1, suggesting both sustained peripheral immune hyperreactivity and a heightened activation state. Taken together, these data indicate that in utero injury primes the immune system and augments enhanced inflammatory signaling. The insidious effects of primed peripheral immune cells may compound PBI secondary to CHORIO and/or placental insufficiency, and thereby render the brain susceptible to future chronic neurological disease. Further understanding of inflammatory mechanisms in PBI may yield clinically important biomarkers and facilitate individualized repair strategies and treatments.

Cognitive development in preterm infants: multifaceted deficits reflect vulnerability of rigorous neurodevelopmental pathways.

Prematurity remains the major cause of neonatal morbidity and mortality, with 15 million preterm births occurring worldwide in 2010. Infants born less than 37 weeks gestation are at high risk of abnormal neurodevelopmental outcomes, given that the central nervous system is extremely sensitive to an abnormal intra- and extra-uterine environment. Children born preterm have multiple neurodevelopmental sequelae involving dynamic and complex cognitive deficits. Former preterm infants have difficulty with each domain of cognition, including executive function, language, learning and memory, complex attention, perceptual-motor function and social cognition when compared to children born at term. Although deficits are not always severe, even mild delays can be impactful, resulting in a spectrum of outcomes from difficulties in school to an inability to lead an independent adult life. Here, we review current literature on the cognitive outcomes of infants born preterm with a focus on how specific disruption in crucial neurodevelopmental pathways render these children vulnerable to dynamic deficits in cognition as they mature. Further, we highlight promising therapies and intervention strategies aimed at mitigating these deficits, including the use of erythropoietin. With an increasing number of preterm infants surviving, understanding developmental deficits will allow therapies to be developed and optimized, in order to ensure the best outcome for this vulnerable patient population.

PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells.

Acid-sensing ion channel 1 (ASIC1) contributes to Ca(2+) influx and contraction in pulmonary arterial smooth muscle cells (PASMC). ASIC1 binds the PDZ (PSD-95/Dlg/ZO-1) domain of the protein interacting with C kinase 1 (PICK1), and this interaction is important for the subcellular localization and/or activity of ASIC1. Therefore, we first hypothesized that PICK1 facilitates ASIC1-dependent Ca(2+) influx in PASMC by promoting plasma membrane localization. Using Duolink to determine protein-protein interactions and a biotinylation assay to assess membrane localization, we demonstrated that the PICK1 PDZ domain inhibitor FSC231 diminished the colocalization of PICK1 and ASIC1 but did not limit ASIC1 plasma membrane localization. Although stimulation of store-operated Ca(2+) entry (SOCE) greatly enhanced colocalization between ASIC1 and PICK1, both FSC231 and shRNA knockdown of PICK1 largely augmented SOCE. These data suggest PICK1 imparts a basal inhibitory effect on ASIC1 Ca(2+) entry in PASMC and led to an alternative hypothesis that PICK1 facilitates the interaction between ASIC1 and negative intracellular modulators, namely PKC and/or the calcium-calmodulin-activated phosphatase calcineurin. FSC231 limited PKC-mediated inhibition of SOCE, supporting a potential role for PICK1 in this response. Additionally, we found PICK1 inhibits ASIC1-mediated SOCE through an effect of calcineurin to dephosphorylate the channel. Furthermore, it appears PICK1/calcineurin-mediated regulation of SOCE opposes PKA phosphorylation and activation of ASIC1. Together our data suggest PKA and PICK1/calcineurin differentially regulate ASIC1-mediated SOCE and these modulatory complexes are important in determining downstream Ca(2+) signaling.

Chronic hypoxia limits H2O2-induced inhibition of ASIC1-dependent store-operated calcium entry in pulmonary arterial smooth muscle.

Our laboratory shows that acid-sensing ion channel 1 (ASIC1) contributes to the development of hypoxic pulmonary hypertension by augmenting store-operated Ca(2+) entry (SOCE) that is associated with enhanced agonist-induced vasoconstriction and arterial remodeling. However, this enhanced Ca(2+) influx following chronic hypoxia (CH) is not dependent on an increased ASIC1 protein expression in pulmonary arterial smooth muscle cells (PASMC). It is well documented that hypoxic pulmonary hypertension is associated with changes in redox potential and reactive oxygen species homeostasis. ASIC1 is a redox-sensitive channel showing increased activity in response to reducing agents, representing an alternative mechanism of regulation. We hypothesize that the enhanced SOCE following CH results from removal of an inhibitory effect of hydrogen peroxide (H2O2) on ASIC1. We found that CH increased PASMC superoxide (O2 (·-)) and decreased rat pulmonary arterial H2O2 levels. This decrease in H2O2 is a result of decreased Cu/Zn superoxide dismutase expression and activity, as well as increased glutathione peroxidase (GPx) expression and activity following CH. Whereas H2O2 inhibited ASIC1-dependent SOCE in PASMC from control and CH animals, addition of catalase augmented ASIC1-mediated SOCE in PASMC from control rats but had no further effect in PASMC from CH rats. These data suggest that, under control conditions, H2O2 inhibits ASIC1-dependent SOCE. Furthermore, H2O2 levels are decreased following CH as a result of diminished dismutation of O2 (·-) and increased H2O2 catalysis through GPx-1, leading to augmented ASIC1-dependent SOCE.

Prenatal Alcohol Exposure and Chorioamnionitis Results in Microstructural Brain Injury in a Preclinical Investigation.

BACKGROUND: Prenatal Alcohol Exposure (PAE) impacts 2% to 5% of infants born in the United States yearly. Women who consume alcohol during pregnancy have a five-fold increased rate of Chorioamnionitis (CHORIO). Both PAE and CHORIO cause microstructural injury to multiple brain regions including major white matter tracts. OBJECTIVE: Utilizing two previously established animal models, we hypothesized that the combination of PAE+CHORIO would result in greater deficits in myelination and structural integrity than PAE alone. MATERIAL AND METHODS: Pregnant Long-Evans rats voluntarily drank 5% ethanol or saccharin until Gestational Day 19 (GD). On GD19, CHORIO was induced in one group of PAE dams by a 30 min uterine artery occlusion and injection of Lipopolysaccharide (LPS) into each amniotic sac. The remaining PAE dams and saccharin controls underwent sham surgery. Pups were born on GD22 and weaned on Postnatal Day 24 (PD). On PD28, offspring were sacrificed, and their brains examined using ex-vivo Diffusion Tensor Imaging (DTI). RESULTS: Compared to control, PAE alone did not affect offspring birth weights, mortality or any DTI measures. In contrast, PAE+CHORIO significantly reduced offspring survival and, in surviving pups, increased Radial Diffusivity (RD) in medial frontal cortex and decreased Fractional Anisotropy (FA) in medial and ventral frontal cortex and within capsular regions. CONCLUSION: The combination of moderate PAE+CHORIO results in an increased mortality, concomitant with diffuse microstructural brain injury noted in young adolescent offspring at PD28. Future studies should examine the extent to which PAE exacerbates the damage caused by CHORIO alone and whether these deficits persist into adulthood.

CXCR2 Blockade Mitigates Neural Cell Injury Following Preclinical Chorioamnionitis.

Minimizing central nervous system (CNS) injury from preterm birth depends upon identification of the critical pathways that underlie essential neurodevelopmental and CNS pathophysiology. While chorioamnionitis (CHORIO), is a leading cause of preterm birth, the precise mechanism linking prenatal brain injury and long-term CNS injury is unknown. The chemokine (C-X-C motif) ligand 1 (CXCL1) and its cognate receptor, CXCR2, are implicated in a variety of uterine and neuropathologies, however, their role in CNS injury associated with preterm birth is poorly defined. To evaluate the putative efficacy of CXCR2 blockade in neural repair secondary to CHORIO, we tested the hypothesis that transient postnatal CXCR2 antagonism would reduce neutrophil activation and mitigate cerebral microstructural injury in rats. To this end, a laparotomy was performed on embryonic day 18 (E18) in Sprague Dawley rats, with uterine arteries transiently occluded for 60 min, and lipopolysaccharide (LPS, 4 µg/sac) injected into each amniotic sac. SB225002, a CXCR2 antagonist (3 mg/kg), was administered intraperitoneally from postnatal day 1 (P1)-P5. Brains were collected on P7 and P21 and analyzed with western blot, immunohistochemistry and ex vivo diffusion tensor imaging (DTI). Results demonstrate that transient CXCR2 blockade reduced cerebral neutrophil activation (myeloperoxidase expression/MPO) and mitigated connexin43 expression, indicative of reduced neuroinflammation at P7 (p < 0.05 for all). CXCR2 blockade also reduced alpha II-spectrin calpain-mediated cleavage, improved pNF/NF ratio, and minimized Iba1 and GFAP expression consistent with improved neuronal and axonal health and reduced gliosis at P21. Importantly, DTI revealed diffuse white matter injury and decreased microstructural integrity following CHORIO as indicated by lower fractional anisotropy (FA) and elevated radial diffusivity (RD) in major white matter tracts (p < 0.05). Early postnatal CXCR2 blockade also reduced microstructural abnormalities in white matter and hippocampus at P21 (p < 0.05). Together, these data indicate that transient postnatal blockade of CXCR2 ameliorates perinatal abnormalities in inflammatory signaling, and facilitates neural repair following CHORIO. Further characterization of neuroinflammatory signaling, specifically via CXCL1/CXCR2 through the placental-fetal-brain axis, may clarify stratification of brain injury following preterm birth, and improve use of targeted interventions in this highly vulnerable patient population.

Evidence for Sexual Dimorphism in the Response to TLR3 Activation in the Developing Neonatal Mouse Brain: A Pilot Study.

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4-6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4-6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p < 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p < 0.001) and IL-10 gene downregulation (-42%, p < 0.05). At 24 h after poly I:C, IL-1ß, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings.

Repetitive Neonatal Erythropoietin and Melatonin Combinatorial Treatment Provides Sustained Repair of Functional Deficits in a Rat Model of Cerebral Palsy.

Cerebral palsy (CP) is the leading cause of motor impairment for children worldwide and results from perinatal brain injury (PBI). To test novel therapeutics to mitigate deficits from PBI, we developed a rat model of extreme preterm birth (<28 weeks of gestation) that mimics dual intrauterine injury from placental underperfusion and chorioamnionitis. We hypothesized that a sustained postnatal treatment regimen that combines the endogenous neuroreparative agents erythropoietin (EPO) and melatonin (MLT) would mitigate molecular, sensorimotor, and cognitive abnormalities in adults rats following prenatal injury. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague-Dawley rats. Uterine artery occlusion was performed for 60 min to induce placental insufficiency via transient systemic hypoxia-ischemia, followed by intra-amniotic injections of lipopolysaccharide, and laparotomy closure. On postnatal day 1 (P1), approximately equivalent to 30 weeks of gestation, injured rats were randomized to an extended EPO + MLT treatment regimen, or vehicle (sterile saline) from P1 to P10. Behavioral assays were performed along an extended developmental time course (n = 6-29). Open field testing shows injured rats exhibit hypermobility and disinhibition and that combined neonatal EPO + MLT treatment repairs disinhibition in injured rats, while EPO alone does not. Furthermore, EPO + MLT normalizes hindlimb deficits, including reduced paw area and paw pressure at peak stance, and elevated percent shared stance after prenatal injury. Injured rats had fewer social interactions than shams, and EPO + MLT normalized social drive. Touchscreen operant chamber testing of visual discrimination and reversal shows that EPO + MLT at least partially normalizes theses complex cognitive tasks. Together, these data indicate EPO + MLT can potentially repair multiple sensorimotor, cognitive, and behavioral realms following PBI, using highly translatable and sophisticated developmental testing platforms.

Preclinical chorioamnionitis dysregulates CXCL1/CXCR2 signaling throughout the placental-fetal-brain axis.

In the United States, perinatal brain injury (PBI) is a major cause of infant mortality and childhood disability. For a large proportion of infants with PBI, central nervous system (CNS) injury begins in utero with inflammation (chorioamnionitis/CHORIO) and/or hypoxia-ischemia. While studies show CHORIO contributes to preterm CNS injury and is also a common independent risk factor for brain injury in term infants, the molecular mechanisms mediating inflammation in the placental-fetal-brain axis that result in PBI remain a gap in knowledge. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been clinically implicated in CHORIO and in mature CNS injury, although their specific role in PBI pathophysiology is poorly defined. Given CXCL1/CXCR2 signaling is essential to neural cell development and neutrophil recruitment, a key pathological hallmark of CHORIO, we hypothesized CHORIO would upregulate CXCL1/CXCR2 expression in the placenta and fetal circulation, concomitant with increased CXCL1/CXCR2 signaling in the developing brain, immune cell activation, neutrophilia, and microstructural PBI. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague Dawley rats to induce CHORIO. Specifically, uterine arteries were occluded for 60min to induce placental transient systemic hypoxia-ischemia (TSHI), followed by intra-amniotic injection of lipopolysaccharide (LPS). Pups were born at E22. Placentae, serum and brain were collected along an extended time course from E19 to postnatal day (P)15 and analyzed using multiplex electrochemiluminescence (MECI), Western blot, qPCR, flow cytometry (FC) and diffusion tensor imaging (DTI). Results demonstrate that compared to sham, CHORIO increases placental CXCL1 and CXCR2 mRNA levels, concomitant with increased CXCR2+ neutrophils. Interestingly, pup serum CXCL1 expression in CHORIO parallels this increase, with sustained elevation through P15. Analyses of CHORIO brains reveal similarly increased CXCL1/CXCR2 expression through P7, together with increased neutrophilia, microgliosis and peripheral macrophages. Similar to the placenta, cerebral neutrophilia was defined by increased CXCR2 surface expression and elevated myeloperoxidase expression (MPO), consistent with immune cell activation. Evaluation of microstructural brain injury at P15 with DTI reveals aberrant microstructural integrity in the callosal and capsular white matter, with reduced fractional anisotropy in superficial and deep layers of overlying cortex. In summary, using an established model of CHORIO that exhibits mature CNS deficits mimicking those of preterm survivors, we show CHORIO induces injury throughout the placental-fetal-brain axis with a CXCL1/CXCR2 inflammatory signature, neutrophilia, and microstructural abnormalities. These data are concomitant with abnormal cerebral CXCL1/CXCR2 expression, and support temporal aberrations in CXCL1/CXCR2 and neutrophil dynamics in the placental-fetal-brain axis following CHORIO. These investigations define novel targets for directed therapies for infants at high risk for PBI.

Neonatal erythropoietin mitigates impaired gait, social interaction and diffusion tensor imaging abnormalities in a rat model of prenatal brain injury.

Children who are born preterm are at risk for encephalopathy of prematurity, a leading cause of cerebral palsy, cognitive delay and behavioral disorders. Current interventions are limited and none have been shown to reverse cognitive and behavioral impairments, a primary determinant of poor quality of life for these children. Moreover, the mechanisms of perinatal brain injury that result in functional deficits and imaging abnormalities in the mature brain are poorly defined, limiting the potential to target interventions to those who may benefit most. To determine whether impairments are reversible after a prenatal insult, we investigated a spectrum of functional deficits and diffusion tensor imaging (DTI) abnormalities in young adult animals. We hypothesized that prenatal transient systemic hypoxia-ischemia (TSHI) would induce multiple functional deficits concomitant with reduced microstructural white and gray matter integrity, and tested whether these abnormalities could be ameliorated using postnatal erythropoietin (EPO), an emerging neurorestorative intervention. On embryonic day 18 uterine arteries were transiently occluded for 60min via laparotomy. Shams underwent anesthesia and laparotomy for 60min. Pups were born and TSHI pups were randomized to receive EPO or vehicle via intraperitoneal injection on postnatal days 1 to 5. Gait, social interaction, olfaction and open field testing was performed from postnatal day 25-35 before brains underwent ex vivo DTI to measure fractional anisotropy, axial diffusivity and radial diffusivity. Prenatal TSHI injury causes hyperactivity, impaired gait and poor social interaction in young adult rats that mimic the spectrum of deficits observed in children born preterm. Collectively, these data show for the first time in a model of encephalopathy of prematurity that postnatal EPO treatment mitigates impairments in social interaction, in addition to gait deficits. EPO also normalizes TSHI-induced microstructural abnormalities in fractional anisotropy and radial diffusivity in multiple regions, consistent with improved structural integrity and recovery of myelination. Taken together, these results show behavioral and memory deficits from perinatal brain injury are reversible. Furthermore, resolution of DTI abnormalities may predict responsiveness to emerging interventions, and serve as a biomarker of CNS injury and recovery.

Extended Erythropoietin Treatment Prevents Chronic Executive Functional and Microstructural Deficits Following Early Severe Traumatic Brain Injury in Rats.

Survivors of infant traumatic brain injury (TBI) are prone to chronic neurological deficits that impose lifelong individual and societal burdens. Translation of novel interventions to clinical trials is hampered in part by the lack of truly representative preclinical tests of cognition and corresponding biomarkers of functional outcomes. To address this gap, the ability of a high-dose, extended, post-injury regimen of erythropoietin (EPO, 3000U/kg/dose × 6d) to prevent chronic cognitive and imaging deficits was tested in a postnatal day 12 (P12) controlled-cortical impact (CCI) model in rats, using touchscreen operant chambers and regional analysis of diffusion tensor imaging (DTI). Results indicate that EPO prevents functional injury and MRI injury after infant TBI. Specifically, subacute DTI at P30 revealed widespread microstructural damage that is prevented by EPO. Assessment of visual discrimination on a touchscreen operant chamber platform demonstrated that all groups can perform visual discrimination. However, CCI rats treated with vehicle failed to pass reversal learning, and perseverated, in contrast to sham and CCI-EPO rats. Chronic DTI at P90 showed EPO treatment prevented contralateral white matter and ipsilateral lateral prefrontal cortex damage. This DTI improvement correlated with cognitive performance. Taken together, extended EPO treatment restores executive function and prevents microstructural brain abnormalities in adult rats with cognitive deficits in a translational preclinical model of infant TBI. Sophisticated testing with touchscreen operant chambers and regional DTI analyses may expedite translation and effective yield of interventions from preclinical studies to clinical trials. Collectively, these data support the use of EPO in clinical trials for human infants with TBI.

Extended Combined Neonatal Treatment With Erythropoietin Plus Melatonin Prevents Posthemorrhagic Hydrocephalus of Prematurity in Rats.

Posthemorrhagic hydrocephalus of prematurity (PHHP) remains a global challenge. Early preterm infants (<32 weeks gestation), particularly those exposed to chorioamnionitis (CAM), are prone to intraventricular hemorrhage (IVH) and PHHP. We established an age-appropriate, preclinical model of PHHP with progressive macrocephaly and ventriculomegaly to test whether non-surgical neonatal treatment could modulate PHHP. We combined prenatal CAM and postnatal day 1 (P1, equivalent to 30 weeks human gestation) IVH in rats, and administered systemic erythropoietin (EPO) plus melatonin (MLT), or vehicle, from P2 to P10. CAM-IVH rats developed progressive macrocephaly through P21. Macrocephaly was accompanied by ventriculomegaly at P5 (histology), and P21 (ex vivo MRI). CAM-IVH rats showed impaired performance of cliff aversion, a neonatal neurodevelopmental test. Neonatal EPO+MLT treatment prevented macrocephaly and cliff aversion impairment, and significantly reduced ventriculomegaly. EPO+MLT treatment prevented matted or missing ependymal motile cilia observed in vehicle-treated CAM-IVH rats. EPO+MLT treatment also normalized ependymal yes-associated protein (YAP) mRNA levels, and reduced ependymal GFAP-immunolabeling. Vehicle-treated CAM-IVH rats exhibited loss of microstructural integrity on diffusion tensor imaging, which was normalized in EPO+MLT-treated CAM-IVH rats. In summary, combined prenatal systemic inflammation plus early postnatal IVH caused progressive macrocephaly, ventriculomegaly and delayed development of cliff aversion reminiscent of PHHP. Neonatal systemic EPO+MLT treatment prevented multiple hallmarks of PHHP, consistent with a clinically viable, non-surgical treatment strategy.

Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension is associated with a decreased antioxidant capacity. However, neither the contribution of reactive oxygen species to pulmonary vasoconstrictor sensitivity, nor the therapeutic efficacy of antioxidant strategies in this setting are known. We hypothesized that reactive oxygen species play a central role in mediating both vasoconstrictor and arterial remodeling components of severe pulmonary arterial hypertension. We examined the effect of the chemical antioxidant, TEMPOL, on right ventricular systolic pressure, vascular remodeling, and enhanced vasoconstrictor reactivity in both chronic hypoxia and hypoxia/SU5416 rat models of pulmonary hypertension. SU5416 is a vascular endothelial growth factor receptor antagonist and the combination of chronic hypoxia/SU5416 produces a model of severe pulmonary arterial hypertension with vascular plexiform lesions/fibrosis that is not present with chronic hypoxia alone. The major findings from this study are: 1) compared to hypoxia alone, hypoxia/SU5416 exposure caused more severe pulmonary hypertension, right ventricular hypertrophy, adventitial lesion formation, and greater vasoconstrictor sensitivity through a superoxide and Rho kinase-dependent Ca2+ sensitization mechanism. 2) Chronic hypoxia increased medial muscularization and superoxide levels, however there was no effect of SU5416 to augment these responses. 3) Treatment with TEMPOL decreased right ventricular systolic pressure in both hypoxia and hypoxia/SU5416 groups. 4) This effect of TEMPOL was associated with normalization of vasoconstrictor responses, but not arterial remodeling. Rather, medial hypertrophy and adventitial fibrotic lesion formation were more pronounced following chronic TEMPOL treatment in hypoxia/SU5416 rats. Our findings support a major role for reactive oxygen species in mediating enhanced vasoconstrictor reactivity and pulmonary hypertension in both chronic hypoxia and hypoxia/SU5416 rat models, despite a paradoxical effect of antioxidant therapy to exacerbate arterial remodeling in animals with severe pulmonary arterial hypertension in the hypoxia/SU5416 model.