Sildenafil improves hippocampal brain injuries and restores neuronal development after neonatal hypoxia–ischemia in male rat pups

AbstractThe hippocampus is a fundamental structure of the brain that plays an important role in neurodevelopment and is very sensitive to hypoxia–ischemia (HI). The purpose of this study was to investigate the effects of sildenafil on neonatal hippocampal brain injuries resulting from HI, and on neuronal development in this context. HI was induced in male Long–Evans rat pups at postnatal day 10 (P10) by a left common carotid ligation followed by a 2-h exposure to 8% oxygen. Rat pups were randomized to vehicle or sildenafil given orally twice daily for 7 days starting 12 h after HI. Hematoxylin and eosin staining was performed at P30 to measure the surface of the hippocampus; immunohistochemistry was performed to stain neurons, oligodendrocytes, and glial cells in the hippocampus. Western blots of the hippocampus were performed at P12, P17, and P30 to study the expression of neuronal markers and mTOR pathway. HI caused significant hippocampal atrophy and a significant reduction of the number of mature neurons, and induced reactive astrocytosis and microgliosis in the hippocampus. HI increased apoptosis and caused significant dysregulation of the normal neuronal development program. Treatment with sildenafil preserved the gross morphology of the hippocampus, reverted the number of mature neurons to levels comparable to sham rats, significantly increased both the immature and mature oligodendrocytes, and significantly reduced the number of microglia and astrocytes. Sildenafil also decreased apoptosis and reestablished the normal progression of post-natal neuronal development. The PI3K/Akt/mTOR pathway, whose activity was decreased after HI in the hippocampus, and restored after sildenafil treatment, may be involved. Sildenafil may have both neuroprotective and neurorestorative properties in the neonatal hippocampus following HI.

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Sildenafil Improves Brain Injury Recovery following Term Neonatal Hypoxia-Ischemia in Male Rat Pups

Developmental Neuroscience ◽

10.1159/000448327 ◽

2016 ◽

Vol 38 (4) ◽

pp. 251-263 ◽

Cited By ~ 2

Author(s):

Armin Yazdani ◽

Zehra Khoja ◽

Aaron Johnstone ◽

Laura Dale ◽

Emmanouil Rampakakis ◽

...

Keyword(s):

Brain Injury ◽

Gait Analysis ◽

Left Hemisphere ◽

Neurological Deficits ◽

Male Rat ◽

Boundary Zone ◽

Neonatal Hypoxia ◽

Rat Pups ◽

Hypoxia Ischemia ◽

Injury Recovery

Term asphyxiated newborns remain at risk of developing brain injury despite available neuropreventive therapies such as hypothermia. Neurorestorative treatments may be an alternative. This study investigated the effect of sildenafil on brain injury induced by neonatal hypoxia-ischemia (HI) at term-equivalent age. Neonatal HI was induced in male Long-Evans rat pups at postnatal day 10 (P10) by left common carotid ligation followed by a 2-hour exposure to 8% oxygen; sham-operated rat pups served as the control. Both groups were randomized to oral sildenafil or vehicle twice daily for 7 consecutive days. Gait analysis was performed on P27. At P30, the rats were sacrificed, and their brains were extracted. The surfaces of both hemispheres were measured on hematoxylin and eosin-stained brain sections. Mature neurons and endothelial cells were quantified near the infarct boundary zone using immunohistochemistry. HI caused significant gait impairment and a reduction in the size of the left hemisphere. Treatment with sildenafil led to an improvement in the neurological deficits as measured by gait analysis, as well as an improvement in the size of the left hemisphere. Sildenafil, especially at higher doses, also caused a significant increase in the number of neurons near the infarct boundary zone. In conclusion, sildenafil administered after neonatal HI may improve brain injury recovery by promoting neuronal populations.

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Gender Differences Involved in the Pathophysiology of the Perinatal Hypoxic-Ischemic Damage

Physiological Research ◽

10.33549/physiolres.934356 ◽

2019 ◽

pp. S207-S217

Author(s):

S. MURDEN ◽

V. BORBÉLYOVÁ ◽

Z. LAŠTŮVKA ◽

J. MYSLIVEČEK ◽

J. OTÁHAL ◽

...

Keyword(s):

Gender Differences ◽

Perinatal Asphyxia ◽

Experimental Studies ◽

Male Rat ◽

Female Rat ◽

Long Term Effects ◽

Rat Pups ◽

Hypoxia Ischemia ◽

Long Term Consequences

Hypoxic-ischemic encephalopathy (HIE) is a neonatal condition that occurs as a consequence of perinatal asphyxia, which is caused by a number of factors, commonly via compression of the umbilical cord, placental abruption, severe meconium aspiration, congenital cardiac or pulmonary anomalies and birth trauma. Experimental studies have confirmed that male rat pups show a higher resistance to HIE treatment. Moreover, the long-term consequences of hypoxia in male are more severe in comparison to female rat pups. These sex differences can be attributed to the pathophysiology of hypoxia-ischemia, whereby studies are beginning to establish such gender-specific distinctions. The current and sole treatment for HIE is hypothermia, in which a reduction in temperature prevents long-term effects, such as cerebral palsy or seizures. However, in most cases hypothermia is not a sufficient treatment as indicated by a high mortality rate. In the present review, we discuss the gender differences within the pathophysiology of hypoxia-ischemia and delve into the role of gender in the incidence, progression and severity of the disease. Furthermore, this may result in the development of potential novel treatment approaches for targeting and preventing the long-term consequences of HIE.

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Regulation of Mineralocorticoid Receptor Expression during Neuronal Differentiation of Murine Embryonic Stem Cells

Endocrinology ◽

10.1210/en.2009-0753 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2244-2254 ◽

Cited By ~ 19

Author(s):

Mathilde Munier ◽

Geri Meduri ◽

Say Viengchareun ◽

Philippe Leclerc ◽

Damien Le Menuet ◽

...

Keyword(s):

Neuronal Differentiation ◽

Mineralocorticoid Receptor ◽

Fluorescent Protein ◽

Morphological Changes ◽

Critical Role ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Receptor Expression ◽

Neuronal Markers ◽

Mature Neurons

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, β-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

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DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature neurons

The Journal of Cell Biology ◽

10.1083/jcb.201804207 ◽

2018 ◽

Vol 218 (1) ◽

pp. 125-133 ◽

Cited By ~ 6

Author(s):

Nathaniel Noblett ◽

Zilu Wu ◽

Zhao Hua Ding ◽

Seungmee Park ◽

Tony Roenspies ◽

...

Keyword(s):

Axonal Regeneration ◽

Neuronal Development ◽

Adult Life ◽

Progressive Increase ◽

Binding Domain ◽

Neuronal Morphology ◽

Loss Of Function ◽

Neuron Morphology ◽

Interacting Protein ◽

Mature Neurons

Neuronal morphology and circuitry established during early development must often be maintained over the entirety of animal lifespans. Compared with neuronal development, the mechanisms that maintain mature neuronal structures and architecture are little understood. The conserved disco-interacting protein 2 (DIP2) consists of a DMAP1-binding domain and two adenylate-forming domains (AFDs). We show that the Caenorhabditis elegans DIP-2 maintains morphology of mature neurons. dip-2 loss-of-function mutants display a progressive increase in ectopic neurite sprouting and branching during late larval and adult life. In adults, dip-2 also inhibits initial stages of axon regeneration cell autonomously and acts in parallel to DLK-1 MAP kinase and EFA-6 pathways. The function of DIP-2 in maintenance of neuron morphology and in axon regrowth requires its AFD domains and is independent of its DMAP1-binding domain. Our findings reveal a new conserved regulator of neuronal morphology maintenance and axon regrowth after injury.

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Experimental neonatal hypoxia ischemia causes long lasting changes of oxidative stress parameters in the hippocampus and the spleen

Journal of Perinatal Medicine ◽

10.1515/jpm-2017-0070 ◽

2018 ◽

Vol 46 (4) ◽

pp. 433-439 ◽

Cited By ~ 8

Author(s):

Felipe Kawa Odorcyk ◽

Janaína Kolling ◽

Eduardo Farias Sanches ◽

Angela T.S. Wyse ◽

Carlos Alexandre Netto

Keyword(s):

Oxidative Stress ◽

Wistar Rat ◽

Mortality And Morbidity ◽

Neonatal Hypoxia ◽

Oxidative Stress Parameters ◽

Rat Pups ◽

Hypoxia Ischemia ◽

Anti Oxidant ◽

Enzyme Superoxide Dismutase ◽

Stress Parameters

Abstract Neonatal hypoxia ischemia (HI) is the main cause of mortality and morbidity in newborns. The mechanisms involved in its progression start immediately and persist for several days. Oxidative stress and inflammation are determinant factors of the severity of the final lesion. The spleen plays a major part in the inflammatory response to HI. This study assessed the temporal progression of HI-induced alterations in oxidative stress parameters in the hippocampus, the most affected brain structure, and in the spleen. HI was induced in Wistar rat pups in post-natal day 7. Production of reactive oxygen species (ROS), and the activity of the anti oxidant enzyme superoxide dismutase and catalase were assessed 24 h, 96 h and 38 days post-HI. Interestingly, both structures showed a similar pattern, with few alterations in the production of ROS species up to 96 h often combined with an increased activity of the anti oxidant enzymes. However, 38 days after the injury, ROS were at the highest in both structures, coupled with a decrease in the activity of the enzymes. Altogether, present results suggest that HI causes long lasting alterations in the hippocampus as well as in the spleen, suggesting a possible target for delayed treatments for HI.

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Does Levetiracetam Administration Prevent Cardiac Damage in Adulthood Rats Following Neonatal Hypoxia/Ischemia-Induced Brain Injury?

Medicina ◽

10.3390/medicina54020012 ◽

2018 ◽

Vol 54 (2) ◽

pp. 12 ◽

Cited By ~ 1

Author(s):

Serkan Gurgul ◽

Belgin Buyukakilli ◽

Mustafa Komur ◽

Cetin Okuyaz ◽

Ebru Balli ◽

...

Keyword(s):

Neonatal Period ◽

Myocardial Damage ◽

Control Group ◽

Protective Effects ◽

Ventricular Contractility ◽

Cardiac Damage ◽

Artery Ligation ◽

Neuroprotective Effects ◽

Rat Pups ◽

Hypoxia Ischemia

Cardiovascular abnormalities are widespread when a newborn is exposed to a hypoxic-ischemic injury in the neonatal period. Although the neuroprotective effects of levetiracetam (LEV) have been reported after hypoxia, the cardioprotective effects of LEV have not been documented. Therefore, we aimed to investigate whether levetiracetam (LEV) has a protective effect on cardiac-contractility and ultrastructure of heart muscle in rats exposed to hypoxia-ischemia (HI) during the neonatal period. A total of 49 seven-day-old rat pups were separated into four groups. For HI induction, a combination of right common carotid artery ligation with 8% oxygen in seven-day-old rat pups for 2 h was performed for saline, LEV100, and LEV200 groups. Just after hypoxia, LEV100 and LEV200 groups were administered with 100 mg/kg and 200 mg/kg of LEV, respectively. The arteries of rats in the control group were only detected; no ligation or hypoxia was performed. At the end of the 16th week after HI, cardiac mechanograms were recorded, and samples of tissue were explored by electronmicroscopy.While ventricular contractility in the control group was similar to LEV100, there were significant decreases in both saline and LEV200 groups (p < 0.05). Although ventricular contractile duration of the control and saline groups was found to be similar, durations in the LEV100 and LEV200 groups were significantly higher (p < 0.05). After HI, mitochondrial damage and ultrastructural deteriorative alterations in ventricles and atriums of the LEV-administered groups were significantly less severe than the saline group. The present study showed that neonatal HI caused long-term cardiac dysfunction and ultrastructural deteriorations in cardiac muscles. LEV administration just after HI might possess some protective effects against myocardial damage and contractility.

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Effect of hyperbaric oxygen on apoptosis in neonatal hypoxia-ischemia rat model

Journal of Applied Physiology ◽

10.1152/japplphysiol.00630.2003 ◽

2003 ◽

Vol 95 (5) ◽

pp. 2072-2080 ◽

Cited By ~ 59

Author(s):

John W. Calvert ◽

Changman Zhou ◽

Anil Nanda ◽

John H. Zhang

Keyword(s):

Hyperbaric Oxygen ◽

Caspase 3 ◽

Apoptotic Cell ◽

Neuroprotective Effect ◽

Parp Cleavage ◽

Artery Ligation ◽

Neonatal Hypoxia ◽

Rat Pups ◽

Hypoxia Ischemia ◽

Expression And Activity

We have previously demonstrated that a transient exposure to hyperbaric oxygen (HBO) attenuated the neuronal injury after neonatal hypoxia-ischemia. This study was undertaken to determine whether HBO offers this neuroprotection by reducing apoptosis in injured brain tissue. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% oxygen). Apoptotic cell death was examined in the injured cortex and hippocampus tissue. Caspase-3 expression and activity increased at 18 and 24 h after the hypoxia-ischemia insult. At 18-48 h, poly(ADP-ribose) polymerase (PARP) cleavage occurred, which reduced the band at 116 kDa and enhanced the band at 85 kDa. There was a time-dependent increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells. A single HBO treatment (100% oxygen, 3 ATA for 1 h) 1 h after hypoxia reduced the enhanced caspase-3 expression and activity, attenuated the PARP cleavage, and decreased the number of TUNEL-positive cells observed in the cortex and hippocampus. These results suggest that the neuroprotective effect of HBO is at least partially mediated by the reduction of apoptosis.

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