A feature of maternal sleep apnea during gestation causes autism-relevant neuronal and behavioral phenotypes in offspring.

Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring's forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment.

Obstructive Sleep Apnea-Associated Intermittent Hypoxia-Induced Immune Responses in Males, Pregnancies, and Offspring.

Obstructive sleep apnea (OSA), a respiratory sleep disorder associated with cardiovascular diseases, is more prevalent in men. However, OSA occurrence in pregnant women rises to a level comparable to men during late gestation, creating persistent effects on both maternal and offspring health. The exact mechanisms behind OSA-induced cardiovascular diseases remain unclear, but inflammation and oxidative stress play a key role. Animal models using intermittent hypoxia (IH), a hallmark of OSA, reveal several pro-inflammatory signaling pathways at play in males, such as TLR4/MyD88/NF-κB/MAPK, miRNA/NLRP3, and COX signaling, along with shifts in immune cell populations and function. Limited evidence suggests similarities in pregnancies and offspring. In addition, suppressing these inflammatory molecules ameliorates IH-induced inflammation and tissue injury, providing new potential targets to treat OSA-associated cardiovascular diseases. This review will focus on the inflammatory mechanisms linking IH to cardiovascular dysfunction in males, pregnancies, and their offspring. The goal is to inspire further investigations into the understudied populations of pregnant females and their offspring, which ultimately uncover underlying mechanisms and therapeutic interventions for OSA-associated diseases.

Gestational intermittent hypoxia increases FosB-immunoreactive perikaryas in the paraventricular nucleus of the hypothalamus of adult male (but not female) rats.

Sleep-disordered breathing is a respiratory disorder commonly experienced by pregnant women. The recurrent hypoxaemic events associated with sleep-disordered breathing have deleterious consequences for the mother and fetus. Adult male (but not female) rats born to dams subjected to gestational intermittent hypoxia (GIH) have a higher resting blood pressure than control animals and show behavioural/neurodevelopmental disorders. The origin of this persistent, sex-specific effect of GIH in offspring is unknown, but disruption of the neuroendocrine stress pathways is a key mechanism by which gestational stress increases disease risk in progeny. Using FosB immunolabelling as a chronic marker of neuronal activation, we determined whether GIH augments basal expression of FosB in the perikaryas of cells in the paraventricular nucleus of the hypothalamus (PVN), a key structure in the regulation of the stress response and blood pressure. From gestational day 10, female rats were subjected to GIH for 8 h/day (light phase) until the day before delivery (gestational day 21); GIH consisted of 2 min hypoxic bouts (10.5% O2 ) alternating with normoxia. Control rats were exposed to intermittent normoxia over the same period (GNX). At adulthood (10-15 weeks), the brains of male and female rats were harvested for FosB immunohistochemistry. In males, GIH augmented PVN FosB labelling density by 30%. Conversely, PVN FosB density in GIH females was 28% lower than that of GNX females. We conclude that GIH has persistent and sex-specific impacts on the development of stress pathways, thereby offering a plausible mechanism by which GIH can disturb neural development and blood pressure homeostasis in adulthood. NEW FINDINGS: What is the central question of this study? In pregnant women, sleep apnoea increases the risk of disease for the offspring at various life stages. Given that gestational stress disrupts the programming of the stress pathways, we determined whether exposing female rats to gestational intermittent hypoxia (GIH) activates hypothalamic neurons regulating the stress response in adult rats. What is the main finding and its importance? Using FosB immunolabelling as a marker of marker of neuronal activation, we showed that GIH augmented basal activation of the paraventricular nucleus of the hypothalamus in males, but not females. Disruption of the stress pathways is a new hypothesis to explain the persistent and sex-specific impacts of GIH on offspring health.

Transcriptional response of murine microglia in Alzheimer's disease and inflammation.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder and is the most common cause of late-onset dementia. Microglia, the primary innate immune cells of the central nervous system (CNS), have a complex role in AD neuropathology. In the initial stages of AD, microglia play a role in limiting pathology by removing amyloid-ß (Aß) by phagocytosis. In contrast, microglia also release pro-inflammatory cytokines and chemokines to promote neuroinflammation and exacerbate AD neuropathology. Therefore, investigating microglial gene networks could identify new targets for therapeutic strategies for AD. RESULTS: We identified 465 differentially expressed genes (DEG) in 5XFAD versus wild-type mice by microarray, 354 DEG in lipopolysaccharide (LPS)-stimulated N9 microglia versus unstimulated control cells using RNA-sequencing (RNA-seq), with 32 DEG common between both datasets. Analyses of the 32 common DEG uncovered numerous molecular functions and pathways involved in Aß phagocytosis and neuroinflammation associated with AD. Furthermore, multiplex ELISA confirmed the induction of several cytokines and chemokines in LPS-stimulated microglia. CONCLUSIONS: In summary, AD triggered multiple signaling pathways that regulate numerous genes in microglia, contributing to Aß phagocytosis and neuroinflammation. Overall, these data identified several regulatory factors and biomarkers in microglia that could be useful in further understanding AD neuropathology.

Silent hypoxaemia in COVID-19 patients.

The clinical presentation of COVID-19 due to infection with SARS-CoV-2 is highly variable with the majority of patients having mild symptoms while others develop severe respiratory failure. The reason for this variability is unclear but is in critical need of investigation. Some COVID-19 patients have been labelled with 'happy hypoxia', in which patient complaints of dyspnoea and observable signs of respiratory distress are reported to be absent. Based on ongoing debate, we highlight key respiratory and neurological components that could underlie variation in the presentation of silent hypoxaemia and define priorities for subsequent investigation.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Sex- and Region-Specific Differences in the Transcriptomes of Rat Microglia from the Brainstem and Cervical Spinal Cord.

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

Nongenomic Actions of 17-ß Estradiol Restore Respiratory Neuroplasticity in Young Ovariectomized Female Rats.

Gonadal steroids modulate CNS plasticity, including phrenic long-term facilitation (pLTF), a form of spinal respiratory neuroplasticity resulting in increased phrenic nerve motor output following exposure to acute intermittent hypoxia (aIH; three 5 min episodes, 10.5% O2). Despite the importance of respiratory system neuroplasticity, and its dependence on estrogen in males, little is known about pLTF expression or mechanisms of estrogen signaling in females. Here, we tested the hypotheses that (1) pLTF expression in young, gonadally intact female rats would be expressed during estrous cycle stages in which 17ß-estradiol (E2) is naturally high (e.g., proestrus vs estrus), (2) pLTF would be absent in ovariectomized (OVX) rats and in physiological conditions in which serum progesterone, but not E2, is elevated (e.g., lactating rats, 3-10 d postpartum), and (3) acute E2 administration would be sufficient to restore pLTF in OVX rats. Recordings of phrenic nerve activity in female Sprague Dawley rats (3-4 months) revealed a direct correlation between serum E2 levels and pLTF expression in cycling female rats. pLTF was abolished with OVX, but was re-established by acute E2 replacement (3 h, intraperitoneal). To identify underlying E2 signaling mechanisms, we intrathecally applied BSA-conjugated E2 over the spinal phrenic motor nucleus and found that pLTF expression was restored within 15 min, suggesting nongenomic E2 effects at membrane estrogen receptors. These data are the first to investigate the role of ovarian E2 in young cycling females, and to identify a role for nongenomic estrogen signaling in any form of respiratory system neuroplasticity.SIGNIFICANCE STATEMENT Exposure to acute intermittent hypoxia induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity that improves breathing in models of spinal cord injury. Although pathways leading to pLTF are well studied in males and estradiol (E2) is known to be required, it has seldom been investigated in females, and underlying mechanisms of E2 signaling are unknown in either sex. We found that while ovariectomy abolished pLTF, it could be restored by acute systemic E2, or by intraspinal application of the membrane-impermeable E2 (BSA-conjugated E2; 15 min). The ability of nongenomic estrogen signaling within the cervical spinal cord to recover respiratory neuroplasticity in disorders of respiratory insufficiency suggests that membrane estrogen receptors may represent novel therapeutic targets to restore breathing in both sexes.

P2Y14 receptor activation decreases interleukin-6 production and glioma GL261 cell proliferation in microglial transwell cultures.

Gliomas are rich in extracellular nucleotides that modulate glioma cell production of multiple cytokines including interleukin (IL)-6, which strongly contributes to glioma cell proliferation. However, little is known about how nucleotide signaling modulates microglial/macrophage (MG/MP) cytokine production in the context of gliomas, nor how MG/MP purinergic P2 receptor expression changes in the tumor micro-environment. We hypothesized that: (1) expression of key P2Y receptors will be augmented in glioma-derived MG/MP, and (2) selective activation of these receptors in vitro will regulate microglial production of IL-6 and glioma cell proliferation. We tested these hypotheses using the murine GL261 glioma model. Compared to MG/MP isolated from the normal brain tissue, CD11b+ cells isolated from GL261 tumors expressed higher levels of several P2 receptors, including P2Y14 receptors. To evaluate microglial P2Y14 receptor function in the context of tumor cells, we first cultured N9 microglia in transwells with GL261 cells and found that microglial P2Y14 mRNA levels were similarly increased in transwell cultures. GL261 cells did not express detectable P2Y14 levels either when they were cultured alone or in transwell cultures with N9 cells. Selective P2Y14 receptor activation with UDP-glucose (UDPG) did not affect IL-6 levels in either cell type cultured alone, but in transwell cultures, UDPG decreased IL-6 protein levels in the medium. Application of conditioned medium from UDPG-treated microglia reduced GL261 cell proliferation. Together, these data suggest that P2Y14 receptors may be a key a receptor involved in glioma cell-MG/MP communication in the tumor environment.

Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration.

BACKGROUND: The bromodomain and extraterminal domain (BET) family proteins (BET2, BET3, and BET4) "read" (bind) histone acetylation marks via two distinct bromodomains (Brom1 and Brom2) facilitating transcriptional activation. These epigenetic "readers" play crucial roles in pathogenic processes such as inflammation. The role of BETs in influencing the degenerative process in the retina is however unknown. METHODS: We employed the rd10 mouse model (Pde6b rd10 mutation) of retinitis pigmentosa (RP) to examine the involvement of BET proteins in retinal neurodegeneration. RESULTS: Inhibition of BET activity by intravitreal delivery of JQ1, a BET-specific inhibitor binding both Brom1 and Brom2, ameliorated photoreceptor degeneration and improved electroretinographic function. Rescue effects of JQ1 were related to the suppression of retinal microglial activation in vivo, as determined by decreased immunostaining of activation markers (IBA1, CD68, TSPO) and messenger RNA (mRNA) levels of inflammatory cytokines in microglia purified from rd10 retinas. JQ1 pre-treatment also suppressed microglial activation in vitro, decreasing microglial proliferation, migration, and mRNA expression of inflammatory cytokines (TNFα, MCP-1, IL-1ß, IL-6, and RANTES). Expression of BET2, but not BET3 and BET4, was significantly elevated during photoreceptor degeneration at postnatal day (PN)24 in retinas of rd10 mice relative to age-matched wild-type controls. siRNA knockdown of BET2 but not BET4, and the inhibitor of Brom2 (RVX208) but not of Brom1 (Olinone), decreased microglial activation. CONCLUSIONS: These findings indicate that BET inhibition rescues photoreceptor degeneration likely via the suppression of microglial activation and implicates BET interference as a potential therapeutic strategy for the treatment of degenerative retinal diseases.

Intermittent Hypoxia-Induced Spinal Inflammation Impairs Respiratory Motor Plasticity by a Spinal p38 MAP Kinase-Dependent Mechanism.

Inflammation is characteristic of most clinical disorders that challenge the neural control of breathing. Since inflammation modulates neuroplasticity, we studied the impact of inflammation caused by prolonged intermittent hypoxia on an important form of respiratory plasticity, acute intermittent hypoxia (three, 5 min hypoxic episodes, 5 min normoxic intervals) induced phrenic long-term facilitation (pLTF). Because chronic intermittent hypoxia elicits neuroinflammation and pLTF is undermined by lipopolysaccharide-induced systemic inflammation, we hypothesized that one night of intermittent hypoxia (IH-1) elicits spinal inflammation, thereby impairing pLTF by a p38 MAP kinase-dependent mechanism. pLTF and spinal inflammation were assessed in anesthetized rats pretreated with IH-1 (2 min hypoxia, 2 min normoxia; 8 h) or sham normoxia and allowed 16 h for recovery. IH-1 (1) transiently increased IL-6 (1.5 ± 0.2-fold; p = 0.02) and inducible nitric oxide synthase (iNOS) (2.4 ± 0.4-fold; p = 0.01) mRNA in cervical spinal homogenates, (2) elicited a sustained increase in IL-1ß mRNA (2.4 ± 0.2-fold; p < 0.001) in isolated cervical spinal microglia, and (3) abolished pLTF (-1 ± 5% vs 56 ± 10% in controls; p < 0.001). pLTF was restored after IH-1 by systemic NSAID administration (ketoprofen; 55 ± 9%; p < 0.001) or spinal p38 MAP kinase inhibition (58 ± 2%; p < 0.001). IH-1 increased phosphorylated (activated) p38 MAP kinase immunofluorescence in identified phrenic motoneurons and adjacent microglia. In conclusion, IH-1 elicits spinal inflammation and impairs pLTF by a spinal p38 MAP kinase-dependent mechanism. By targeting inflammation, we may develop strategies to manipulate respiratory motor plasticity for therapeutic advantage when the respiratory control system is compromised (e.g., sleep apnea, apnea of prematurity, spinal injury, or motor neuron disease).

Age-dependent differences in microglial responses to systemic inflammation are evident as early as middle age.

Whereas age increases microglial inflammatory activities and impairs their ability to effectively regulate their immune response, it is unclear at what age these exaggerated responses begin. We tested the hypotheses that augmented microglial responses to inflammatory challenge are present as early as middle age and that repeated stimulation of primed microglia in vivo would reveal microglial senescence. Microglial gene expression was investigated in a mouse model of repeated systemic inflammation induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS). Following LPS, microglia from middle-aged mice (9-10 mo) displayed larger increases in Tnfα, Il-6, and Il-1ß gene expression compared with young adults (2 mo). Similar results were observed in the spleens of middle-aged mice, indicating that exaggeration of both central and peripheral immune responses are already evident at early middle age. Interestingly, despite greater proinflammatory responses to the first LPS challenge in the aged mice, there were no age-dependent differences in either microglia or spleen following a subsequent LPS dose, suggesting that animals at this age retain the ability to effectively control their immune response following repeated challenge. The exacerbated microglial immune response to systemic inflammation at early middle age suggests that the CNS may be vulnerable to age-dependent alterations earlier than previously appreciated.

Mechanisms of microglial activation in models of inflammation and hypoxia: Implications for chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a hallmark of sleep apnoea, a condition associated with diverse clinical disorders. CIH and sleep apnoea are characterized by increased reactive oxygen species formation, peripheral and CNS inflammation, neuronal death and neurocognitive deficits. Few studies have examined the role of microglia, the resident CNS immune cells, in models of CIH. Thus, little is known concerning their direct contributions to neuropathology or the cellular mechanisms regulating their activities during or following pathological CIH. In this review, we identify gaps in knowledge regarding CIH-induced microglial activation, and propose mechanisms based on data from related models of hypoxia and/or hypoxia-reoxygenation. CIH may directly affect microglia, or may have indirect effects via the periphery or other CNS cells. Peripheral inflammation may indirectly activate microglia via entry of pro-inflammatory molecules into the CNS, and/or activation of vagal afferents that trigger CNS inflammation. CIH-induced release of damage-associated molecular patterns from injured CNS cells may also activate microglia via interactions with pattern recognition receptors expressed on microglia. For example, Toll-like receptors activate mitogen-activated protein kinase/transcription factor pathways required for microglial inflammatory gene expression. Although epigenetic effects from CIH have not yet been studied in microglia, potential epigenetic mechanisms in microglial regulation are discussed, including microRNAs, histone modifications and DNA methylation. Epigenetic effects can occur during CIH, or long after it has ended. A better understanding of CIH effects on microglial activities may be important to reverse CIH-induced neuropathology in patients with sleep disordered breathing.

CSF1 overexpression has pleiotropic effects on microglia in vivo.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 overexpression on microglia, transgenic mice that overexpress CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 overexpressing mice have increased microglial proliferation and increased microglial numbers compared with controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 overexpressing and control mice. Microglia in CSF1 overexpressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide. These results indicate that the CSF1 overexpression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats.

Activation of microglia, CNS resident immune cells, is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons. Despite evidence that microglia contribute to disease progression, the exact role of these cells in ALS pathology remains unknown. We immunomagnetically isolated microglia from different CNS regions of SOD1(G93A) rats at three different points in disease progression: presymptomatic, symptom onset and end-stage. We observed no differences in microglial number or phenotype in presymptomatic rats compared to wild-type controls. Although after disease onset there was no macrophage infiltration, there were significant increases in microglial numbers in the spinal cord, but not cortex. At disease end-stage, microglia were characterized by high expression of galectin-3, osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, BDNF and arginase-1. Flow cytometry revealed the presence of at least two phenotypically distinct microglial populations in the spinal cord. Immunohistochemistry showed that galectin-3/osteopontin positive microglia were restricted to the ventral horns of the spinal cord, regions with severe motor neuron degeneration. End-stage SOD1(G93A) microglia from the cortex, a less affected region, displayed similar gene expression profiles to microglia from wild-type rats, and displayed normal responses to systemic inflammation induced by LPS. On the other hand, end-stage SOD1(G93A) spinal microglia had blunted responses to systemic LPS suggesting that in addition to their phenotypic changes, they may also be functionally impaired. Thus, after disease onset, microglia acquired unique characteristics that do not conform to typical M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was observed only in the most affected CNS regions, suggesting that overexpression of mutated hSOD1 is not sufficient to trigger these changes in microglia. These novel observations suggest that microglial regional and phenotypic heterogeneity may be an important consideration when designing new therapeutic strategies targeting microglia and neuroinflammation in ALS.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations.

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

Acute Postnatal Inflammation Alters Adult Microglial Responses to LPS that Are Sex-, Region- and Timing of Postnatal Inflammation-Dependent.

BACKGROUND: Adverse events in early life can have impact lasting into adulthood. We investigated the long-term effects of systemic inflammation during postnatal development on adult microglial responses to LPS in two CNS regions (cortex, cervical spinal cord) in male and female rats. METHODS: Inflammation was induced in Sprague-Dawley rats by lipopolysaccharide (LPS, 1 mg/kg) administered intraperitoneally during postnatal development at P7, P12 or P18. As adults (12 weeks of age), the rats received a second LPS dose (1 mg/kg). Control rats received saline. Microglia were isolated 3 hours post-LPS from the cortex and cervical spinal cord. Gene expression was assessed via qRT-PCR for pro-inflammatory (IL-6, iNOS, Ptgs2, C/EBPb, CD14, CXCL10), anti-inflammatory (CD68, Arg-1), and homeostatic genes (P2Y12, Tmemm119). CSF-1 and CX3CL1 mRNA was analyzed in microglia-free homogenates. RESULTS: Basal gene expression in adult microglia was largely unaffected by early life LPS. Changes in adult microglial pro-inflammatory genes in response to LPS were either unchanged or attenuated in rats exposed to LPS during postnatal development. Ptgs2, C/EBPb, CXCL10 and Arg-1 were the genes most affected, with expression levels significantly downregulated vs control rats without postnatal LPS exposure. Cortical microglia were affected more by postnatal inflammation than spinal microglia, and males were more impacted than females. Overall, inflammatory challenge at P18 had the greatest effect on adult microglial gene expression, whereas challenge at P7 had less impact. Microglial homeostatic genes were unaffected by postnatal LPS. CONCLUSIONS: Long-lasting effects of postnatal inflammation on adult microglia depend on the timing of postnatal inflammation, CNS region and sex.

Gestational Intermittent Hypoxia Enhances Mammary Stem Cells and Alters Tumor Phenotype in Adult Female Offspring.

An adverse perinatal environment can increase long-term cancer risk, although the precise nature of associated perinatal triggers remain unknown. Sleep apnea is a common condition during pregnancy, characterized by recurrent cessations in breathing during sleep, and the potential consequences of sleep apnea during pregnancy as it relates to breast cancer risk in offspring have not been explored. To model sleep apnea, Sprague-Dawley dams were exposed during gestation to nightly intermittent hypoxia (GIH) or normoxia (GNx), and the mammary glands of female offspring were examined. GIH offspring demonstrated increased epithelial stem and progenitor cell populations, which are associated with diminished transforming growth factor beta (TGFß) activity. Elevations in adipose tissue stem cells in the mammary gland were also identified in GIH offspring. In aging females, mammary tumors formed in GIH offspring. These tumors displayed a dramatic increase in stroma compared to tumors from GNx offspring, as well as distinct patterns of expression of stem cell-related pathways. Together, these results suggest that exposure to sleep apnea during pregnancy leads to lasting changes in the mammary glands of female offspring. Increased stem and progenitor cell populations as a result of GIH exposure could enhance long-term breast cancer risk, as well as alter the clinical behavior of resulting breast tumors.