Disruption of Interneuron Neurogenesis in Premature Newborns and Reversal with Estrogen Treatment.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1+, Dlx2+, and Sox2+ cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1+ progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2+ cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1+ and Dlx2+ cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants.SIGNIFICANCE STATEMENT Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants.

Absence of gut microbial colonization attenuates the sympathoadrenal response to hypoglycemic stress in mice: implications for human neonates.

BACKGROUND: Gut microbiota plays an important role during early development via bidirectional gut-brain signaling. Catecholamines provide a survival advantage allowing adaptation to common postnatal stressors. We aimed to explore the potential link between gut microbiota/gut-derived metabolites and sympathoadrenal stress responsivity. METHODS: The effect of insulin-induced hypoglycemia was compared in mice with (control, adapted control) and without microbiome (germ-free, GF). Counter-regulatory hormones were analyzed in urine and plasma. Adrenal gene expression levels were evaluated and correlated to cecal short chain fatty acids (SCFA) content. RESULTS: There was a significant association between absent microbiota/SCFA and epinephrine levels at baseline and after stress. Corticosterone (hypothalamic-pituitary-adrenal axis) and glucagon release (parasympathetic signaling) were similar in all groups. Hypoglycemia-induced c-Fos (marker of trans-synaptic neuronal activation) in both conditions. Delayed increases in adrenal tyrosine hydroxylase and neuropeptide Y messenger RNA were observed in GF mice. Transcriptome analysis provided insight into underlying mechanisms for attenuated epinephrine production and release. CONCLUSION: Lack of microbiome selectively impaired adrenal catecholamine responses to hypoglycemia. We speculate that absent/delayed acquisition of flora (e.g., after antibiotic exposure) may compromise sympathoadrenal stress responsivity. Conversely, controlled manipulation of the intestinal microflora may provide a novel therapeutic opportunity to improve survival and overall health in preterm neonates.

Extended Production of Cortical Interneurons into the Third Trimester of Human Gestation.

In humans, the developmental origins of interneurons in the third trimester of pregnancy and the timing of completion of interneuron neurogenesis have remained unknown. Here, we show that the total and cycling Nkx2.1(+)and Dlx2(+)interneuron progenitors as well as Sox2(+)precursor cells were higher in density in the medial ganglionic eminence (MGE) compared with the lateral ganglionic eminence and cortical ventricular/subventricular zone (VZ/SVZ) of 16-35 gw subjects. The proliferation of these progenitors reduced as a function of gestational age, almost terminating by 35 gw. Proliferating Dlx2(+)cells were higher in density in the caudal ganglionic eminence (CGE) compared with the MGE, and persisted beyond 35 gw. Consistent with these findings, Sox2, Nkx2.1, Dlx2, and Mash1 protein levels were higher in the ganglionic eminences relative to the cortical VZ/SVZ. The density of gamma-aminobutyric acid-positive (GABA(+)) interneurons was higher in the cortical VZ/SVZ relative to MGE, but Nkx2.1 or Dlx2-expressing GABA(+)cells were more dense in the MGE compared with the cortical VZ/SVZ. The data suggest that the MGE and CGE are the primary source of cortical interneurons. Moreover, their generation continues nearly to the end of pregnancy, which may predispose premature infants to neurobehavioral disorders.

Epidermal growth factor preserves myelin and promotes astrogliosis after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) leads to reduced myelination and astrogliosis of the white matter in premature infants. No therapeutic strategy exists to minimize white matter injury in survivors with IVH. Epidermal growth factor (EGF) enhances myelination, astrogliosis, and neurologic recovery in animal models of white matter injury. Here, we hypothesized that recombinant human (rh) EGF treatment would enhance oligodendrocyte precursor cell (OPC) maturation, myelination, and neurological recovery in preterm rabbits with IVH. In addition, rhEGF would promote astrogliosis by inducing astroglial progenitor proliferation and GFAP transcription. We tested these hypotheses in a preterm rabbit model of IVH and evaluated autopsy samples from human preterm infants. We found that EGF and EGFR expression were more abundant in the ganglionic eminence relative to the cortical plate and white matter of human infants and that the development of IVH reduced EGF levels, but not EGFR expression. Accordingly, rhEGF treatment promoted proliferation and maturation of OPCs, preserved myelin in the white matter, and enhanced neurological recovery in rabbits with IVH. rhEGF treatment inhibited Notch signaling, which conceivably contributed to OPC maturation. rhEGF treatment contributed to astrogliosis by increasing astroglial proliferation and upregulating GFAP as well as Sox9 expression. Hence, IVH results in a decline in EGF expression; and rhEGF treatment preserves myelin, restores neurological recovery, and exacerbates astrogliosis by inducing proliferation of astrocytes and enhancing transcription of GFAP and Sox9 in pups with IVH. rhEGF treatment might improve the neurological outcome of premature infants with IVH. GLIA 2016;64:1987-2004.

Neurogenesis continues in the third trimester of pregnancy and is suppressed by premature birth.

Premature infants exhibit neurodevelopmental delay and reduced growth of the cerebral cortex. However, the underlying mechanisms have remained elusive. Therefore, we hypothesized that neurogenesis in the ventricular and subventricular zones of the cerebral cortex would continue in the third trimester of pregnancy and that preterm birth would suppress neurogenesis. To test our hypotheses, we evaluated autopsy materials from human fetuses and preterm infants of 16-35 gestational weeks (gw). We noted that both cycling and noncycling Sox2(+) radial glial cells and Tbr2(+) intermediate progenitors were abundant in human preterm infants until 28 gw. However, their densities consistently decreased from 16 through 28 gw. To determine the effect of premature birth on neurogenesis, we used a rabbit model and compared preterm [embryonic day 29 (E29), 3 d old] and term (E32, <2 h old) pups at an equivalent postconceptional age. Glutamatergic neurogenesis was suppressed in preterm rabbits, as indicated by the reduced number of Tbr2(+) intermediate progenitors and the increased number of Sox2(+) radial glia. Additionally, hypoxia-inducible factor-1α, vascular endothelial growth factor, and erythropoietin were higher in term than preterm pups, reflecting the hypoxic intrauterine environment of just-born term pups. Proneural genes, including Pax6 and Neurogenin-1 and -2, were higher in preterm rabbit pups compared with term pups. Importantly, neurogenesis and associated factors were restored in preterm pups by treatment with dimethyloxallyl glycine, a hypoxia mimetic agent. Hence, glutamatergic neurogenesis continues in the premature infants, preterm birth suppresses neurogenesis, and hypoxia-mimetic agents might restore neurogenesis, enhance cortical growth, and improve neurodevelopmental outcome of premature infants.

Treatment with thyroxine restores myelination and clinical recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRß. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

Angiogenic inhibition reduces germinal matrix hemorrhage.

The germinal matrix of premature infants is selectively vulnerable to hemorrhage within the first 48 h of life. To assess the role of vascular immaturity in germinal matrix hemorrhage (GMH), we evaluated germinal matrix angiogenesis in human fetuses and premature infants, as well as in premature rabbit pups, and noted active vessel remodeling in all three. Vascular endothelial growth factor (VEGF), angiopoietin-2 and endothelial cell proliferation were present at consistently higher levels in the germinal matrix relative to the white matter anlagen and cortical mantle. On that basis, we asked whether prenatal treatment with either of two angiogenic inhibitors, the COX-2 inhibitor celecoxib, or the VEGFR2 inhibitor ZD6474, could suppress the incidence of GMH in premature rabbit pups. Celecoxib treatment decreased angiopoietin-2 and VEGF levels as well as germinal matrix endothelial proliferation. Furthermore, treatment with celecoxib or ZD6474 substantially decreased the incidence of GMH. Thus, by suppressing germinal matrix angiogenesis, prenatal celecoxib or ZD6474 treatment may be able to reduce both the incidence and severity of GMH in susceptible premature infants.

Effect of acetate supplementation on traumatic stress-induced behavioral impairments in male rats.

Gut microbiota and their metabolites have emerged as key players in the pathogenesis of neuropsychiatric disorders. Recently, we demonstrated that animals susceptible to Single Prolonged Stress (SPS) have an overall pro-inflammatory gut microbiota and significantly lower cecal acetate levels than SPS-resilient rats, which correlated inversely with the anxiety index. Here, we investigated whether the microbial metabolite, acetate, could ameliorate SPS-triggered impairments. Male rats were randomly divided into unstressed controls or groups exposed to SPS. The groups received continued oral supplementation of either 150 mM of sodium acetate or 150 mM of sodium chloride-matched water. Two weeks after SPS, a battery of behavioral tests was performed, and the animals were euthanized the following day. While not affecting the unstressed controls, acetate supplementation reduced the impact of SPS on body weight gain and ameliorated SPS-induced anxiety-like behavior and the impairments in social interaction, but not depressive-like behavior. These changes were accompanied by several beneficial effects of acetate supplementation. Acetate alleviated the stress response by reducing urinary epinephrine levels, induced epigenetic modification by decreasing histone deacetylase (HDAC2) gene expression, inhibited neuroinflammation by reducing the density of Iba1+ cells and the gene expression of IL-1ß in the hippocampus, and increased serum ß-hydroxybutyrate levels. The findings reveal a causal relationship between oral acetate treatment and mitigation of several SPS-induced behavioral impairments. Mechanistically, it impacted neuronal and metabolic pathways including changes in stress response, epigenetic modifications, neuroinflammation and showed novel link to ketone body production. The study demonstrates the preventive-therapeutic potential of acetate supplementation to alleviate adverse responses to traumatic stress.

Bibliometric analysis of worldwide research trends on breast cancer about inflammation.

Background: The most prevalent cancer and the second-leading cause of cancer-related mortality in women is breast cancer. Growing interest has been shown in recent years in learning more about the processes behind the development of breast cancer. It has been shown that persistent inflammation may play a significant role in the advancement of breast cancer. However, a comprehensive and objective analysis on the state of inflammation in breast cancer research is still lacking. This study was aim to undertake a bibliometric analysis of breast cancer research associated with inflammation between 2013 and 2022 in order to identify the trends, dynamics, and scientific outputs in the field. Methods: From 2013 to 2022, original and review publications on breast cancer and inflammation-associated research were retrieved from the Web of Science Core Collection (WOSCC) database. To examine the position of yearly publications, journals, nations, institutions, and authors, we employed two bibliometric tools (CiteSpace and VOSviewer). After that, by examining keyword visualization and keyword bursts, we determined the hot research fields related to inflammation in breast cancer. Results: we discovered 6902 publications regarding inflammation in breast cancer by using our retrieval approach. In terms of the number of publications, The United States ranked first in the global study, followed by China and Italy. In terms of institutions, the University of Texas System, UT MD Anderson Cancer Center, and University of California System are in the top 3 for the quantity of publications published. The most popular journal for this field research is "CANCERS." Ueno NT, Woodward WA, Cristofanilli M, and others have made significant contributions to the understanding of inflammation in breast cancer. In the end, we conducted a biclustering analysis on keywords and discovered three clusters that represent research hotspots. Conclusion: According to the global trend, the research output of inflammation in breast cancer is increasing. The information provided in this article, including the cooperation network information of authors, nations, journals, and institutions, may help researchers to better understand hotspots and developing patterns in this discipline. At present, the focus of study gradually shifts from "phenotype study" to "therapeutic research". It is recommended to pay attention to the latest hot spots, such as targeted therapy, antimicrobial activity and nanoparticle.

Differences in gut microbiota associated with stress resilience and susceptibility to single prolonged stress in female rodents.

Exposure to traumatic stress is a major risk factor for the development of neuropsychiatric disorders in a subpopulation of individuals, whereas others remain resilient. The determinants of resilience and susceptibility remain unclear. Here, we aimed to characterize the microbial, immunological, and molecular differences between stress-susceptible and stress-resilient female rats before and after exposure to a traumatic experience. Animals were randomly divided into unstressed controls (n = 10) and experimental groups (n = 16) exposed to Single Prolonged Stress (SPS), an animal model of PTSD. Fourteen days later, all rats underwent a battery of behavioral tests and were sacrificed the following day to collect different organs. Stool samples were collected before and after SPS. Behavioral analyses revealed divergent responses to SPS. The SPS treated animals were further subdivided into SPS-resilient (SPS-R) and SPS-susceptible (SPS-S) subgroups. Comparative analysis of fecal 16S sequencing before and after SPS exposure indicated significant differences in the gut microbial composition, functionality, and metabolites of the SPS-R and SPS-S subgroups. In line with the observed distinct behavioral phenotypes, the SPS-S subgroup displayed higher blood-brain barrier permeability and neuroinflammation relative to the SPS-R and/or controls. These results indicate, for the first time, pre-existing and trauma-induced differences in the gut microbial composition and functionality of female rats that relate to their ability to cope with traumatic stress. Further characterization of these factors will be crucial for understanding susceptibility and fostering resilience, especially in females, who are more likely than males to develop mood disorders.

Early Postnatal Expression of Tgfß-1 and Fgf-2 Correlates With Regenerative Functions of Unrestricted Somatic Stem Cell Infusion After Rabbit GMH-IVH.

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth associated with white matter injury (WMI) and reduced neurogenesis. IVH commonly arises from the germinal matrix, a highly cellular, transient structure, where all precursor cells are born, proliferate, and migrate during brain development. IVH leads to reduced progenitor cell proliferation and maturation and contributes to WMI. Interruption of oligodendrocyte lineage (OL) proliferation and maturation after IVH will prevent myelination. We evaluated whether unrestricted somatic stem cells (USSCs) could recover OL lineage, as USSC release multiple relevant growth factors and cytokines. The effects of USSC infusion at 24 hours after IVH were assessed in the periventricular zone by analysis of OL lineage-specific progression (PDGFR+, OLIG2+, NKX2.2+ with Ki67), and this was correlated with growth factors TGFß1, FGF2 expression. The early OL cell lineage by immunofluorescence and cell density quantitation showed significant reduction after IVH (P < .05 both PDGFR+, OLIG2+ at day 3); with significant recovery after injection of USSCs (P < .05 both PDGFR+, OLIG2+ at day 3). CSF protein and tissue mRNA levels of TGFß1 were reduced by IVH and recovered after USSC (P < .05 for all changes). FGF2 showed an increased mRNA after USSC on day3 (P < .05). Cell cyclin genes were unaffected except for the cycle inhibitor P27Kip1 which increased after IVH but returned to normal after USSC on day 3. Our findings demonstrated a plausible mechanism through which USSCs can aid in developmental myelination by recovery of OL proliferation and maturation along with correlative changes in growth factors during brain development.

Bone morphogenetic protein inhibition promotes neurological recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in neural cell death and white matter injury in premature infants. No therapeutic strategy is currently available against this disorder. Bone morphogenetic protein (BMP) signaling suppresses oligodendrocyte development through basic-helix-loop-helix (bHLH) transcription factors and promotes astrocytosis. Therefore, we hypothesized that IVH in premature newborns initiates degeneration and maturation arrest of oligodendrocyte lineage and that BMP inhibition alleviates hypomyelination, gliosis, and motor impairment in the survivors of IVH. To test the hypotheses, a rabbit model of IVH was used in which premature rabbit pups (E29) are treated with intraperitoneal glycerol at 2 h of age to induce IVH; and the pups with IVH exhibit hypomyelination and gliosis at 2 weeks of postnatal age. Maturation of oligodendrocyte lineage was evaluated by specific markers, and the expression of bHLH transcription factors was assessed. BMP levels were measured in both premature rabbit pups and autopsy materials from premature infants. Recombinant human noggin was used to suppress BMP action; and neurobehavioral performance, myelination and gliosis were assessed in noggin-treated pups compared with untreated controls. We found that IVH resulted in apoptosis and reduced proliferation of oligodendrocyte progenitors, as well as arrested maturation of preoligodendrocytes in rabbits. BMP4 levels were significantly elevated in both rabbit pups and human premature infants with IVH compared with controls. Importantly, BMP inhibition by recombinant human noggin restored the levels of phospho-Smad1/5/8, Olig2 transcription factor, oligodendrocyte maturation, myelination, astrocyte morphology, and motor function in premature pups with IVH. Hence, BMP inhibition might enhance neurological recovery in premature infants with IVH.

Novel organotypic in vitro slice culture model for intraventricular hemorrhage of premature infants.

Mechanisms of brain injury in intraventricular hemorrhage (IVH) of premature infants are elusive, and no therapeutic strategy exists to prevent brain damage in these infants. Therefore, we developed an in vitro organotypic forebrain slice culture model to advance mechanistic studies and therapeutic developments for this disorder. We cultured forebrain slices from E29 rabbit pups and treated the cultured slices (CS) with moderate (50 µl) or large (100 µl) amounts of autologous blood to mimic moderate and severe IVH. Blood-induced damage to CS was evaluated by propidium iodide staining, lactate dehydrogenase (LDH) levels, microglial density, neuronal degeneration, myelination, and gliosis over 2 weeks after the initiation of culture. CS were viable for at least 14 days in vitro (DIV). The application of blood induced significant neural cell degeneration. Degenerating cells were more abundant and LDH levels were elevated in a dose-dependent manner in CS treated with 50 versus 100 µl of blood compared with untreated controls. Microglial density was higher in blood-treated CS compared with controls at both 7 and 14 days posttreatment, and myelination was reduced and gliosis enhanced. Selective application of blood fractions revealed that CS treated with plasma displayed more hypomyelination and gliosis compared with erythrocyte-treated slices. This study develops and characterizes a novel rabbit forebrain slice culture model of IVH that exhibits neuropatholgical changes similar to those in human infants with IVH. Importantly, plasma appears to induce greater white matter damage than erythrocytes in IVH,indicating plasma as a source of neurotoxic components.

Neuroprotection in a rabbit model of intraventricular haemorrhage by cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-alpha inhibition.

Intraventricular haemorrhage is a major complication of prematurity that results in neurological dysfunctions, including cerebral palsy and cognitive deficits. No therapeutic options are currently available to limit the catastrophic brain damage initiated by the development of intraventricular haemorrhage. As intraventricular haemorrhage leads to an inflammatory response, we asked whether cyclooxygenase-2, its derivative prostaglandin E2, prostanoid receptors and pro-inflammatory cytokines were elevated in intraventricular haemorrhage; whether their suppression would confer neuroprotection; and determined how cyclooxygenase-2 and cytokines were mechanistically-linked. To this end, we used our rabbit model of intraventricular haemorrhage where premature pups, delivered by Caesarian section, were treated with intraperitoneal glycerol at 2 h of age to induce haemorrhage. Intraventricular haemorrhage was diagnosed by head ultrasound at 6 h of age. The pups with intraventricular haemorrhage were treated with inhibitors of cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α; and cell-infiltration, cell-death and gliosis were compared between treated-pups and vehicle-treated controls during the first 3 days of life. Neurobehavioural performance, myelination and gliosis were assessed in pups treated with cyclooxygenase-2 inhibitor compared to controls at Day 14. We found that both protein and messenger RNA expression of cyclooxygenase-2, prostaglandin E2, prostanoid receptor-1, tumour necrosis factor-α and interleukin-1ß were consistently higher in the forebrain of pups with intraventricular haemorrhage relative to pups without intraventricular haemorrhage. However, cyclooxygenase-1 and prostanoid receptor 2-4 levels were comparable in pups with and without intraventricular haemorrhage. Cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α inhibition reduced inflammatory cell infiltration, apoptosis, neuronal degeneration and gliosis around the ventricles of pups with intraventricular haemorrhage. Importantly, cyclooxygenase-2 inhibition alleviated neurological impairment, improved myelination and reduced gliosis at 2 weeks of age. Cyclooxygenase-2 or prostanoid receptor-1 inhibition reduced tumour necrosis factor-α level, but not interleukin-1ß. Conversely, tumour necrosis factor-α antagonism did not affect cyclooxygenase-2 expression. Hence, prostanoid receptor-1 and tumour necrosis factor-α are downstream to cyclooxygenase-2 in the inflammatory cascade induced by intraventricular haemorrhage, and cyclooxygenase-2-inhibition or suppression of downstream molecules--prostanoid receptor-1 or tumour necrosis factor-α--might be a viable neuroprotective strategy for minimizing brain damage in premature infants with intraventricular haemorrhage.

Bacteria - derived short chain fatty acids restore sympathoadrenal responsiveness to hypoglycemia after antibiotic-induced gut microbiota depletion.

The microbiome co-evolved with their mammalian host over thousands of years. This commensal relationship serves a pivotal role in various metabolic, physiological, and immunological processes. Recently we discovered impaired adrenal catecholamine stress responses in germ-free mice suggesting developmental modification of the reflex arc or absence of an ongoing microbiome signal. To determine whether maturational arrest or an absent bacteria-derived metabolite was the cause, we tested whether depleting gut microbiome in young adult animals could also alter the peripheral stress responses to insulin-induced hypoglycemia. Groups of C57Bl6 male mice were given regular water (control) or a cocktail of non-absorbable broad-spectrum antibiotics (Abx) in the drinking water for two weeks before injection with insulin or saline. Abx mice displayed a profound decrease in microbial diversity and abundance of Bacteroidetes and Firmicutes, plus a markedly enlarged caecum and no detectable by-products of bacterial fermentation (sp. short chain fatty acids, SCFA). Tonic and stress-induced epinephrine levels were attenuated. Recolonization (Abx + R) restored bacterial diversity, but not the sympathoadrenal system responsiveness or caecal acetate, propionate and butyrate levels. In contrast, corticosterone (HPA) and glucagon (parasympathetic) resting values and responses to hypoglycemia remained similar across all conditions. Oral supplementation with SCFA improved epinephrine responses to hypoglycaemia. Whole genome shotgun sequence profiling of fecal samples from control, Abx and Abx + R cohorts identified nine microbes (SCFA producers) absent from both Abx and Abx + R groups. These results implicate gut microbiome depletion plus its attendant reduction in SCFA signalling in adversely affecting the release of epinephrine in response to hypoglycemia. We speculate that regardless of postnatal age, a mutable microbiome messaging system exists throughout life. Unravelling these mechanisms could lead to new therapeutic possibilities through controlled manipulation of the gut microbiota and its ability to alter systemic neurotransmitter responsiveness.

Human Cord Blood Derived Unrestricted Somatic Stem Cells Restore Aquaporin Channel Expression, Reduce Inflammation and Inhibit the Development of Hydrocephalus After Experimentally Induced Perinatal Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth associated with cerebral palsy, intellectual disability, and commonly, accumulation of cerebrospinal fluid (CSF). Histologically, IVH leads to subependymal gliosis, fibrosis, and disruption of the ependymal wall. Importantly, expression of aquaporin channels 1 and 4 (AQP1 and AQP4) regulating respectively, secretion and absorption of cerebrospinal fluids is altered with IVH and are associated with development of post hemorrhagic hydrocephalus. Human cord blood derived unrestricted somatic stem cells (USSCs), which we previously demonstrated to reduce the magnitude of hydrocephalus, as having anti-inflammatory, and beneficial behavioral effects, were injected into the cerebral ventricles of rabbit pups 18 h after glycerol-induced IVH. USSC treated IVH pups showed a reduction in ventricular size when compared to control pups at 7 and 14 days (both, P < 0.05). Histologically, USSC treatment reduced cellular infiltration and ependymal wall disruption. In the region of the choroid plexus, immuno-reactivity for AQP1 and ependymal wall AQP4 expression were suppressed after IVH but were restored following USSC administration. Effects were confirmed by analysis of mRNA from dissected choroid plexus and ependymal tissue. Transforming growth factor beta (TGF-ß) isoforms, connective tissue growth factor (CTGF) and matrix metalloprotease-9 (MMP-9) mRNA, as well as protein levels, were significantly increased following IVH and restored towards normal with USSC treatment (P < 0.05). The anti-inflammatory cytokine Interleukin-10 (IL-10) mRNA was reduced in IVH, but significantly recovered after USSC injection (P < 0.05). In conclusion, USSCs exerted anti-inflammatory effects by suppressing both TGF-ß specific isoforms, CTGF and MMP-9, recovered IL-10, restored aquaporins expression towards baseline, and reduced hydrocephalus. These results support the possibility of the use of USSCs to reduce IVH consequences in prematurity.

Effect of prenatal glucocorticoids on cerebral vasculature of the developing brain.

BACKGROUND AND PURPOSE: Prenatal glucocorticoids prevent germinal matrix hemorrhage in premature infants. The underlying mechanism, however, is elusive. Germinal matrix is enriched with angiogenic vessels exhibiting paucity of pericytes and glial fibrillary acidic protein-positive astrocyte end feet. Therefore, we asked whether glucocorticoid treatment would suppress angiogenesis and enhance periendothelial coverage by pericytes and glial fibrillary acidic protein-positive end feet in the germinal matrix microvasculature. METHODS: We treated pregnant rabbits with intramuscular betamethasone and delivered pups prematurely by cesarean section at E29 (term=32 days). Endothelial turnover, vascular density, pericyte coverage, glial fibrillary acidic protein-positive end feet, cell death, and growth factors orchestrating angiogenesis, including vascular endothelial growth factor, angiopoietins, transforming growth factor-beta, and platelet-derived growth factor-B, were compared between betamethasone-treated and untreated pups. Similar comparisons were done between autopsy materials from premature infants exposed and unexposed to prenatal glucocorticoids. RESULTS: Antenatal glucocorticoid treatment reduced endothelial proliferation, vascular density, and vascular endothelial growth factor expression in the germinal matrix of both rabbits and humans. The pericyte coverage was greater in glucocorticoid-treated rabbit pups and human infants than in controls, but not the glial fibrillary acidic protein-positive end feet coverage. Transforming growth factor-beta, but not angiopoietins and platelet-derived growth factor-B, were elevated in glucocorticoid-treated rabbit pups compared with controls. Betamethasone treatment induced apoptosis, neuronal degeneration, and gliosis in rabbit pups. However, there was no evidence of increased cell death in glucocorticoid-exposed human infants. CONCLUSIONS: Prenatal glucocorticoid suppresses vascular endothelial growth factor and elevates transforming growth factor-beta levels, which results in angiogenic inhibition, trimming of neovasculature, and enhanced pericyte coverage. These changes contribute to stabilizing the germinal matrix vasculature, thereby reducing its propensity to hemorrhage. Prenatal glucocorticoid exposure does not induce neural cell death in humans, unlike rabbits.

Development of integrins in the vasculature of germinal matrix, cerebral cortex, and white matter of fetuses and premature infants.

Germinal matrix (GM) vasculature is selectively vulnerable to hemorrhage in premature infants during the first 48 hr of life. This is attributed to rapid angiogenesis of this brain region, resulting in formation of nascent vessels that show a paucity of pericytes and immaturity of extracellular matrix. Integrins are key regulators of angiogenesis and contribute to stabilization of cerebral vasculature by providing endothelial- and astrocyte-matrix adhesion. Therefore, we asked whether GM exhibited a distinct regional pattern of integrin expression that was dissimilar from that of the cerebral cortex and white matter in human fetuses and premature infants. To this end, we measured protein and gene expression of integrins in the GM, cortex, and white matter of human fetuses (15-22 weeks), premature infants (23-35 weeks), and mature infants (36-40 weeks). We found that protein levels of alpha5beta1 integrin were greater in the GM than in the cortex or white matter by 1.6-fold for both fetuses and premature infants. alpha5beta1 integrin mRNA expression was higher in the GM than in the cortex or white matter by 2-fold for fetuses but not for premature infants. alphaVbeta3, alphaVbeta5, alphaVbeta8, and alpha4beta1 integrin expression were comparable among GM, cortex, and white matter in fetuses and premature infants. Because alpha5beta1 integrin is a central regulator of angiogenesis, its elevation in the GM of fetuses and premature infants indicates that this might be a key activator of endothelial proliferation in this brain region. We speculate that selective alpha5beta1 integrin inhibition might suppress angiogenesis in the GM and thus prevent brain hemorrhage in premature infants.

A central role of connexin 43 in hypoxic preconditioning.

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

Oxidative-nitrosative stress in a rabbit pup model of germinal matrix hemorrhage: role of NAD(P)H oxidase.

BACKGROUND AND PURPOSE: Germinal matrix hemorrhage-intraventricular hemorrhage is the most common neurological problem of premature infants. Despite this, mechanisms of brain injury from intraventricular hemorrhage are elusive. We hypothesized that germinal matrix hemorrhage-intraventricular hemorrhage, by induction of NAD(P)H oxidases, might cause oxidative/nitrosative stress contributing to brain injuries and that NAD(P)H oxidase inhibition could offer neuroprotection. METHODS: To test this hypothesis, we exploited our rabbit pup model of glycerol-induced germinal matrix hemorrhage-intraventricular hemorrhage. We delivered rabbit pups prematurely (E29) by cesarean section and administered intraperitoneal glycerol at 2 hours postnatal age. Free-radical adducts, including nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine as well as O(2)(.-) and H(2)O(2) levels were measured in the forebrain. To determine the source of free-radical generation, we used inhibitors for NAD(P)H oxidase (apocynin), xanthine oxidase (allopurinol), cyclo-oxygenase-2 (indomethacin), or nitric oxide synthases (L-NAME). Intraventricular hemorrhage pups were treated with apocynin and cell death was compared between apocynin-treated and vehicle-treated pups. RESULTS: Nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine levels were higher in pups with intraventricular hemorrhage than controls. Likewise, O(2)(.-) and H(2)O(2) levels were significantly greater in both the periventricular area and cerebral cortex of pups with intraventricular hemorrhage than controls. In pups with intraventricular hemorrhage, reactive oxygen species production was more in the periventricular area than in the cortex. Apocynin, but not allopurinol, indomethacin, or nitric oxide synthases, inhibited reactive oxygen species generation. Importantly, apocynin reduced cell death in pups with intraventricular hemorrhage. CONCLUSIONS: Activation of NAD(P)H oxidase was the predominant mechanism of free-radical generation in pups with intraventricular hemorrhage. NAD(P)H oxidase inhibition by apocynin might suppress reactive oxygen species production and confer neuroprotection in premature infants with intraventricular hemorrhage.