Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

Objective

To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage.

Methods

A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3.

Results

Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro.

Interpretation

These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults.     

Dexamethasone Protects Neonatal Hypoxic-Ischemic Brain Injury via L-PGDS-Dependent PGD2-DP1-pERK Signaling Pathway

Background and Purpose

Glucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D₂ (PGD₂)-DP₁-pERK mediated signaling pathway.

Methods

Dexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD₂, D prostanoid (DP₁) receptor, pERK1/2 and PGD₂ were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups.

Results

Dexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD₂. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl₄), DP₁ (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains.

Conclusion

Interaction of glucocorticoids-GR signaling and L-PGDS-PGD₂-DP₁-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury.     

Attenuated AMPA Receptor Expression Allows Glioblastoma Cell Survival in Glutamate-Rich Environment

Background

Glioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na⁺ and Ca²⁺-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment.

Methods and Findings

In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited.

Conclusions

Together with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumors.     

Organ Dysfunction among Piglets Treated with Inhaled Nitric Oxide and Intravenous Hydrocortisone during Prolonged Endotoxin Infusion

Objective

It has previously been shown that a combination of inhaled nitric oxide (iNO) and intravenous (IV) steroid attenuates endotoxin-induced organ damage in a 6-hour porcine endotoxemia model. We aimed to further explore these effects in a 30-hour model with attention to clinically important variables.

Design

Randomized controlled trial.

Setting

University animal laboratory.

Subjects

Domestic piglets (n = 30).

Interventions

Animals were randomized into 5 groups (n = 6 each): 1) Controls, 2) LPS-only (endotoxin/lipopolysaccharide (LPS) infusion), 3) LPS + iNO, 4) LPS + IV steroid, 5) LPS + iNO + IV steroid.

Measurements and Main Results

Exposure to LPS temporarily increased pulmonary artery mean pressure and impeded renal function with elevated serum creatinine and acidosis compared to a control group over the 30-hour study period. Double treatment with both iNO and IV steroid tended to blunt the deterioration in renal function, although the only significant effect was on Base Excess (p = 0.045). None of the LPS + iNO + IV steroid treated animals died during the study period, whereas one animal died in each of the other LPS-infused groups.

Conclusions

This study suggests that combined early therapy with iNO and IV steroid is associated with partial protection of kidney function after 30 hours of experimental LPS infusion.     

Behavioral Consequences of NMDA Antagonist-Induced Neuroapoptosis in the Infant Mouse Brain

Background

Exposure to NMDA glutamate antagonists during the brain growth spurt period causes widespread neuroapoptosis in the rodent brain. This period in rodents occurs during the first two weeks after birth, and corresponds to the third trimester of pregnancy and several years after birth in humans. The developing human brain may be exposed to NMDA antagonists through drug-abusing mothers or through anesthesia.

Methodology/Principal Findings

We evaluated the long-term neurobehavioral effects of mice exposed to a single dose of the NMDA antagonist, phencyclidine (PCP), or saline, on postnatal day 2 (P2) or P7, or on both P2 and P7. PCP treatment on P2 + P7 caused more severe cognitive impairments than either single treatment. Histological examination of acute neuroapoptosis resulting from exposure to PCP indicated that the regional pattern of degeneration induced by PCP in P2 pups was different from that in P7 pups. The extent of damage when evaluated quantitatively on P7 was greater for pups previously treated on P2 compared to pups treated only on P7.

Conclusions

These findings signify that PCP induces different patterns of neuroapoptosis depending on the developmental age at the time of exposure, and that exposure at two separate developmental ages causes more severe neuropathological and neurobehavioral consequences than a single treatment.     

Chlorpromazine protects against apoptosis induced by exogenous stimuli in the developing rat brain

Background

Chlorpromazine (CPZ), a commonly used antipsychotic drug, was found to play a neuroprotective role in various models of toxicity. However, whether CPZ has the potential to affect brain apoptosis in vivo is still unknown. The purpose of this study was to investigate the potential effect of CPZ on the apoptosis induced by exogenous stimuli.

Methodology

The ethanol treated infant rat was utilized as a valid apoptotic model, which is commonly used and could trigger robust apoptosis in brain tissue. Prior to the induction of apoptosis by subcutaneous injection of ethanol, 7-day-old rats were treated with CPZ at several doses (5 mg/kg, 10 mg/kg and 20 mg/kg) by intraperitoneal injection. Apoptotic cells in the brain were measured using TUNEL analysis, and the levels of cleaved caspase-3, cytochrome c, the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 were assessed by immunostaining or western blot.

Findings

Compared to the group injected with ethanol only, the brains of the CPZ-pretreated rats had fewer apoptotic cells, lower expression of cleaved caspase-3, cytochrome c and Bax, and higher expression of Bcl-2. These results demonstrate that CPZ could prevent apoptosis in the brain by regulating the mitochondrial pathway.

Conclusions

CPZ exerts an inhibitory effect on apoptosis induced by ethanol in the rat brain, intimating that it may offer a means of protecting nerve cells from apoptosis induced by exogenous stimuli.     

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro

Background

Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.

Results

Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.

Conclusion

Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.     

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Background

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer''s disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and Findings

In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer''s disease mouse model.

Conclusions

Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.     

Decreased GABAB receptor function in the cerebellum and brain stem of hypoxic neonatal rats: Role of glucose,oxygen and epinephrine resuscitation

Background-

Hypoxia during the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. The neurobiological changes mediated through neurotransmitters and other signaling molecules associated with neonatal hypoxia are an important aspect in establishing a proper neonatal care.

Methods-

The present study evaluated total GABA, GABA_B receptor alterations, gene expression changes in GABA_B receptor and glutamate decarboxylase in the cerebellum and brain stem of hypoxic neonatal rats and the resuscitation groups with glucose, oxygen and epinephrine. Radiolabelled GABA and baclofen were used for receptor studies of GABA and GABA_B receptors respectively and Real Time PCR analysis using specific probes for GABA_B receptor and GAD mRNA was done for gene expression studies.

Results-

The adaptive response of the body to hypoxic stress resulted in a reduction in total GABA and GABA_B receptors along with decreased GABA_B receptor and GAD gene expression in the cerebellum and brain stem. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations.

Conclusions-

Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation, which helps in encountering hypoxia. The present study suggests that reduction in the GABA_B receptors functional regulation during hypoxia plays an important role in central nervous system damage. Resuscitation with glucose alone and glucose and oxygen to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Intrinsic Up-Regulation of 2-AG Favors an Area Specific Neuronal Survival in Different In Vitro Models of Neuronal Damage

Background

The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).

Results

2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.

Conclusion

Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Nitric Oxide Administration Using an Oxygen Hood: A Pilot Trial

Background

We have shown earlier that inhaled nitric oxide (iNO) administered by oxygen hood reduces pulmonary hypertension in an animal model (J Perinatol 2002; 22:50-6). Our objective in this study was to determine feasibility of iNO by oxygen hood in neonates with elevated alveolar-arterial oxygen gradients (A-aDO₂).

Methods/Principal Findings

Masked randomized controlled pilot trial. Inclusion criteria were: gestation≥34 weeks, age<7 days, with post-ductal arterial line, and A-aDO₂ 400–600. Infants were randomized to study gas (iNO 20 ppm or equivalent O₂ flow) for 1 hr which was then weaned over the next 4 hours. Primary outcome was PaO₂ one hour post-randomization. Four infants each were randomized to iNO or O₂ (controls). Two of the four infants given iNO had an increase in PaO₂ of >100 torr, while oxygenation was unchanged in the controls. Methemoglobinemia and other adverse effects were not noted in any infant. Environmental levels of NO and NO₂ were minimal (<1 ppm) at >0.3 m from the hood.

Conclusions

Administration of iNO by oxygen hood is feasible. Larger randomized controlled trials are required to measure the efficacy and determine an appropriate target population for this technique.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00041548","term_id":"NCT00041548"}}NCT00041548     

Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model

Introduction

Branched chain fatty acids (BCFA) are found in the normal term human newborn''s gut, deposited as major components of vernix caseosa ingested during late fetal life. We tested the hypothesis that premature infants'' lack of exposure to gastrointestinal (GI) BCFA is associated with their microbiota and risk for necrotizing enterocolitis (NEC) using a neonatal rat model.

Methods

Pups were collected one day before scheduled birth. The pups were exposed to asphyxia and cold stress to induce NEC. Pups were assigned to one of three experimental treatments. DF (dam-fed) ; Control, hand-fed rat milk substitute ; BCFA, hand-fed rat milk substitute with 20%w/w BCFA. Total fat was equivalent (11%wt) for both the Control and BCFA groups. Cecal microbiota were characterized by 16S rRNA gene pyrosequencing, and intestinal injury, ileal cytokine and mucin gene expression, interleukin-10 (IL-10) peptide immunohistochemistry, and BCFA uptake in ileum phospholipids, serum and liver were assessed.

Results

NEC incidence was reduced by over 50% in the BCFA group compared to the Control group as assessed in ileal tissue; microbiota differed among all groups. BCFA-fed pups harbored greater levels of BCFA-associated Bacillus subtilis and Pseudomonas aeruginosa compared to Controls. Bacillus subtilis levels were five-fold greater in healthy pups compared to pups with NEC. BCFA were selectively incorporated into ileal phospholipids, serum and liver tissue. IL-10 expression increased three-fold in the BCFA group versus Controls and no other inflammatory or mucosal mRNA markers changed.

Conclusion

At constant dietary fat level, BCFA reduce NEC incidence and alter microbiota composition. BCFA are also incorporated into pup ileum where they are associated with enhanced IL-10 and may exert other specific effects.     

Inhibition of Phosphatase and Tensin Homolog Deleted on Chromosome 10 Decreases Rat Cortical Neuron Injury and Blood-Brain Barrier Permeability,and Improves Neurological Functional Recovery in Traumatic Brain Injury Model

Background and Purpose

Recent evidence has supported the neuroprotective effect of bpV (pic), an inhibitor of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), in models of ischemic stroke. However, whether PTEN inhibitors improve long-term functional recovery after traumatic brain injury (TBI) and whether PTEN affects blood brain barrier (BBB) permeability need further elucidation. The present study was performed to address these issues.

Methods

Adult Sprague-Dawley rats were subjected to fluid percussion injury (FPI) after treatment with a well-established PTEN inhibitor bpV (pic) or saline starting 24 h before FPI. Western blotting, real-time quantitative PCR, or immunostaining was used to measure PTEN, p-Akt, or MMP-9 expression. We determined the presence of neuron apoptosis by TUNEL assay. Evans Blue dye extravasation was measured to evaluate the extent of BBB disruption. Functional recovery was assessed by the neurological severity score (NSS), and Kaplan-Meier analysis was used for survival analysis.

Results

PTEN expression was up-regulated after TBI. After bpV (pic) treatment, p-Akt was also up-regulated. We found that bpV (pic) significantly decreased BBB permeability and reduced the number of TUNEL-positive cells. We further demonstrated that PTEN inhibition improved neurological function recovery in the early stage after TBI.

Conclusion

These data suggest that treatment with the PTEN inhibitor bpV (pic) has a neuroprotective effect in TBI rats.     

Expression of Aquaporin 4 and Breakdown of the Blood-Brain Barrier after Hypoglycemia-Induced Brain Edema in Rats

Background

Hypoglycemia-induced brain edema is a severe clinical event that often results in death. The mechanisms by which hypoglycemia induces brain edema are unclear.

Methods

In a hypoglycemic injury model established in adult rats, brain edema was verified by measuring brain water content and visualizing water accumulation using hematoxylin and eosin staining. Temporal expression of aquaporin 4 (AQP4) and the integrity of the blood-brain barrier (BBB) were evaluated. We assessed the distribution and expression of AQP4 following glucose deprivation in astrocyte cultures.

Results

Brain edema was induced immediately after severe hypoglycemia but continued to progress even after recovery from hypoglycemia. Upregulation of AQP4 expression and moderate breakdown of the BBB were observed 24 h after recovery. In vitro, significant redistribution of AQP4 to the plasma membrane was induced following 6 h glucose deprivation.

Conclusion

Hypoglycemia-induced brain edema is caused by cytotoxic and vasogenic factors. Changes in AQP4 location and expression may play a protective role in edema resolution.     

Nicotinamide inhibits alkylating agent-induced apoptotic neurodegeneration in the developing rat brain

Background

Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain.

Methodology/Principal Findings

Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment.

Conclusion

Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.     

Obesity Induced by Neonatal Overfeeding Worsens Airway Hyperresponsiveness and Inflammation

Background

Obesity is a risk factor for the development of certain respiratory diseases, and neonatal overfeeding results in an early onset of obesity in adulthood. However, the influence of neonatal overfeeding on respiratory diseases has rarely been studied. Therefore, this paper is aimed at investigating the effect of neonatal overfeeding on airway responsiveness and inflammation.

Methodology/Principal Findings

The neonatal overfeeding was induced by reducing litter size to three pups per litter (small litter, SL) in contrast to the normal litter size with ten pups per litter (NL) on postnatal day 3 (P3) in male ICR mice. On P21, mice were weaned to standard chow diet. Airway responsiveness to methacholine was measured either on P21 or P150. Total and classified inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted, lung inflammatory cells were evaluated through staining with hematoxylin & eosin and F4/80 immunohistochemistry; lung fibrosis was evaluated through staining with Masson and α-SAM immunohistochemistry. Leptin levels in serum were measured by RIA; TNF-α levels in serum and BALF were quantified by ELISA; mRNA levels of TNF-α, CTGF and TGF-β1 in lung tissues were measured using real-time PCR. Mice from SL exhibited accelerated body weight gain, impaired glucose tolerance and hyperleptinemia. Enhanced airway responsiveness to methacholine was observed in SL mice on P150, but not on P21. Pulmonary inflammation was evident in SL mice on P150, as reflected by inflammatory cells especially macrophages around bronchi and interstitium. BALF and serum TNF-α levels and lung TNF-α mRNA expression were significantly increased in SL mice on P150. More collagen accumulated surrounding the bronchi on P150; lung mRNA levels of TGF-β1 and CTGF were also increased on P150.

Conclusion

In addition to inducing a variety of metabolic defects, neonatal overfeeding enhanced lung inflammation, which may lead to airway remodeling and airway hyperresponsiveness in adulthood.     

Protective effect of a sesamin derivative, 3-bis (3-methoxybenzyl) butane-1, 4-diol on ischemic and hypoxic neuronal injury

Background

Stroke is one of the leading causes of neuronal death. Sesamin is known for neuroprotection by its antioxidant and anti-inflammatory properties but it lacks blood–brain barrier (BBB) activity. A panel of sesamin derivatives was screened and 3-bis (3-methoxybenzyl) butane-1,4-diol (BBD) was selected for high BBB activity and tested for its neuroprotective effect.

Methods

The focal cerebral ischemia of Sprague–Dawley rats and hypoxia models of murine BV-2 microglia or PC12 cells under oxygen/glucose deprivation were used for in vivo and in vitro test, respectively. Lipid peroxidation and superoxide dismutase (SOD) activity from the ischemic brain were tested and reactive oxygen species (ROS), cytokine production, prostaglandin (PGE₂) and related signaling pathways from hypoxic cells were examined by ELISA or Western blot assay, respectively.

Results

BBD showed a protective effect when given 90 min after the focal cerebral ischemia. It also reduced lipid peroxidation and preserved SOD activity from the ischemic brain. The mechanism of BBD was further confirmed by attenuating ROS, cytokine production, and PGE₂ release from hypoxic BV-2 or PC12 cells. BBD significantly reduced hypoxia-induced c-Jun N-terminal kinases (JNK) and modulated AKT-1 and caspase-3 (survival and apoptotic pathways) in BV-2 cells, and inhibited hypoxia-induced JNK and cyclooxygenase-2 activation in PC12 cells.

Conclusions

The neuroprotective effect of BBD on ischemia/hypoxia models was involved with antioxidant and anti-inflammatory effects. The result would help the development of new CNS drug for protection of ischemia/hypoxia injury.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

ABO Blood Group Is Associated with Response to Inhaled Nitric Oxide in Neonates with Respiratory Failure

Background

Inhaled nitric oxide (iNO) reduces death or need for extracorporeal membrane oxygenation (ECMO) in infants with persistent pulmonary hypertension of the newborn (PPHN). However, the response to iNO is variable and only 50–60% of infants demonstrate a response to iNO. It is not known why only some infants respond to iNO. Adults and children with blood groups B or AB do not respond as well to iNO as those with blood groups O/A.

Methods/Principal Findings

To determine if blood group was associated with iNO response in newborn infants, a retrospective medical record review was done of infants admitted to a regional NICU from 2002-9 with a diagnosis of PPHN. Data were collected during the first twelve hours post-initiation of treatment. Of 86 infants diagnosed with PPHN, 23 infants had blood group A [18 received iNO], 21 had group B [18 with iNO], 40 had group O [36 with iNO], and 2 had group AB [both received iNO]. Change in PaO₂/FiO₂ was less in infants with blood group A, of whom less than half were responders (ΔPaO₂/FiO₂>20%) at 12 h versus 90% of infants with either O or B. Race, sex, birth weight, gestational age, Apgar scores at 1 and 5 minutes, and baseline PaO₂/FiO₂ were similar among groups. Outcomes including need for ECMO, death, length of ventilatory support, length of iNO use, and hospital stay were statistically not different by blood groups.

Conclusions/Significance

Our results indicate that blood group influences iNO response in neonates. We hypothesize that either there is genetic linkage of the ABO gene locus with vasoregulatory genes, or that blood group antigens directly affect vascular reactivity.