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Neonatal Mice (neonatal + mouse)
Selected AbstractsMaturation-Dependent Alcohol Resistance in the Developing Mouse: Cerebellar Neuronal Loss and Gene Expression During Alcohol-Vulnerable and -Resistant PeriodsALCOHOLISM, Issue 8 2008Bahri Karaçay Background:, Alcohol abuse during pregnancy injures the fetal brain. One of alcohol's most important neuroteratogenic effects is neuronal loss. Rat models have shown that the cerebellum becomes less vulnerable to alcohol-induced neuronal death as it matures. We determined if maturation-dependent alcohol resistance occurs in mice and compared patterns of gene expression during the alcohol resistant and sensitive periods. Methods:, Neonatal mice received alcohol daily over postnatal day (PD) 2 to 4 or PD8 to 10. Purkinje cells and granule cells were quantified on PD25. The temporal expression patterns of 4 neuro-developmental genes and 3 neuro-protective genes in the cerebellum were determined daily over PD0 to 15 to determine how gene expression changes as the cerebellum transitions from alcohol-vulnerable to alcohol-resistant. The effect of alcohol on expression of these genes was determined when the cerebellum is alcohol sensitive (PD4) and resistant (PD10). Results:, Purkinje and granule cells were vulnerable to alcohol-induced death at PD2 to 4, but not at PD8 to 10. Acquisition of maturation-dependent alcohol resistance coincided with changes in the expression of neurodevelopmental genes. The vulnerability of cerebellar neurons to alcohol toxicity declined in parallel with decreasing levels of Math1 and Cyclin D2, markers of immature granule cells. Likewise, the rising resistance to alcohol toxicity paralleled increasing levels of GABA ,-6 and Wnt-7a, markers of mature granule neurons. Expression of growth factors and genes with survival promoting function (IGF-1, BDNF, and cyclic AMP response element binding protein) did not rise as the cerebellum transitioned from alcohol-vulnerable to alcohol-resistant. All 3 were expressed at substantial levels during the vulnerable period and were not expressed at higher levels later. Acute alcohol exposure altered the expression of neurodevelopmental genes and growth factor genes when administered either during the alcohol vulnerable period or resistant period. However, the patterns in which gene expression changed varied among the genes and depended on timing of alcohol administration. Conclusions:, Mice have a temporal window of vulnerability in the first week of life, during which cerebellar neurons are more sensitive to alcohol toxicity than during the second week. Expression of genes governing neuronal maturation changes in synchrony with the acquisition of alcohol resistance. Growth factors do not rise as the cerebellum transitions from alcohol-vulnerable to alcohol-resistant. Thus, a process intrinsic to neuronal maturation, rather than rising levels of growth factors, likely underlies maturation-dependent alcohol resistance. [source] Selective expression of the small GTPase RhoB in the early developing mouse lensDEVELOPMENTAL DYNAMICS, Issue 3 2001Rupalatha Maddala Abstract This report describes the expression and distribution pattern of RhoB GTPase in the developing mouse lens. RhoB expression was confirmed by sequencing an reverse transcriptase-polymerase chain reaction,generated DNA fragment of RhoB. Immunohistochemical analysis of RhoB revealed expression in the lens vesicle (both anterior and posterior vesicle) at embryonic day (E) 11.5, and in the epithelium and primary fibers of the E14.5 lens. Compared with the neonatal stage (day 1), where RhoB is detected in the entire lens (epithelium, primary, and secondary fibers), expression of this protein is restricted to the epithelial and outer cortical secondary fibers in postnatal lenses (from day 7 to day18). Interestingly, in E11.5 and E14.5 lenses, RhoB is localized predominantly in the lens, but not detectable in the retina, cornea, or other ocular tissues. RhoB expression appears to be down-regulated in the postnatal lens with concomitant up-regulation in the retina and cornea, compared with earlier stages of development (eyes of E11.5, E14.5, and neonatal mice). This study reveals the selective expression of RhoB in the lens during early eye development and suggests a potential role for this small GTPase in cytoskeletal reorganization associated with lens epithelial cell elongation and differentiation. © 2001 Wiley-Liss, Inc. [source] Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor , expressionDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001Christine K. Wagner Abstract Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER,) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER, gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER, in these regions. In contrast, PRir levels in neocortex are not altered by ER, gene disruption. The results of this study suggest that the induction of PR via ER, may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 176,182, 2001 [source] Maternal behavior changes after immune challenge of neonates with developmental effects on adult social behaviorDEVELOPMENTAL PSYCHOBIOLOGY, Issue 1 2003Kathryn E. Hood Abstract To examine whether maternal responsiveness during interactions with endotoxin-treated pups contributes to long-term effects on social development, neonatal mice were fostered on postnatal day 1 to dams from three selectively bred lines that differ in social behaviors. On day 5, neonates were administered saline or 0.5 mg/kg endotoxin (lipopolysaccharide, i.p.). Observations of undisturbed dams and litters on days 2, 4, 6, and 8 showed modest line differences in maternal behaviors. At the peak intensity of the transient illness induced by endotoxin (3 hr postinjection on day 5), dams increased licking and decreased time off-nest for endotoxin, but not saline-treated pups. As adults, fostered-reared males were observed in brief social interactions. Males exposed to endotoxin early in life showed changes in adult social behaviors that depended on foster dam line as well as individual differences in maternal responsiveness. Maternal responsiveness to stressed neonates can ameliorate the social,developmental effects of early illness. © 2003 Wiley Periodicals, Inc. Dev Psychobiol 42: 17,34, 2003. [source] Treatment of neonatal mice with Flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced IL-12 and IFN-, productionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2004Sabine Vollstedt Abstract Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8, and MHC class,II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-,. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T,cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines. [source] Excess target-derived neurotrophin-3 alters the segmental innervation of the skinEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2001Amy M. Ritter Abstract It is thought that dermatomes are established during development as a result of competition between afferents of neighbouring segments. Mice that overexpress neurotrophins in the skin provide an interesting model to test this hypothesis, as they possess increased numbers of sensory neurons, and display hyperinnervation of the skin. When dermatomal boundaries were mapped in adult mice, it was found that those in nerve growth factor and brain-derived neurotrophic factor overexpressers were indistinguishable from wild-type animals but that overlap between adjacent segments was greatly reduced in neurotrophin-3 (NT-3) overexpressers. However, dermatomes in heterozygous NT-3 knockout mice displayed no more overlap than wild-types. In order to quantify differences across strains, innervation territories of thoracic dorsal cutaneous nerves were mapped and measured in adult mice. Overlap between adjacent dorsal cutaneous nerves was normal in nerve growth factor overexpressing mice, but much reduced in NT-3 overexpressers. However, this restriction was not reflected in the central projection of the dorsal cutaneous nerve, creating a mismatch between peripheral and central projections. Dorsal cutaneous nerve territories were also mapped in neonatal mice aged postnatal day 7,8. In neonates, nerve territories of NT-3 overexpressers overlapped less than wild-types, but in neonates of both strains the amount of overlap was much greater than in the adult. These results indicate that substantial separation of dermatomes occurs postnatally, and that excess NT-3 enhances this process, resulting in more restricted dermatomes. It may exert its effects either by enhancing competition, or by direct effects on the stability and formation of sensory endings in the skin. [source] Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal miceGLIA, Issue 8 2009Kai Härtel Abstract A controlled, periodic exchange of air between lungs and atmosphere requires a neuronal rhythm generated by a network of neurons in the ventral respiratory group (VRG) of the brainstem. Glial cells, e.g. astrocytes, have been shown to be supportive in stabilizing this neuronal activity in the central nervous system during development. In addition, a variety of neuromodulators including serotonin (5-HT), Substance P (SP), and thyrotropin-releasing hormone (TRH) stimulate respiratory neurons directly. If astrocytes in the VRG, like their neuronal neighbors, are also directly stimulated by neuromodulators, they might indirectly affect the respiratory neurons and consequently the respiratory rhythm. In the present study, we provide support for this concept by demonstrating expression of NK1-R, TRH-R, and 5-HT2 -R in astrocytes of the VRG with immunohistochemistry. Additionally, we showed that the external application of the neuromodulators 5-HT, SP, and TRH activate calcium transients in VRG astrocytes. Consequently, we postulate that in the VRG of the neonatal mouse, neuromodulation by SP, TRH, and serotonin also involves astrocytic calcium signaling. © 2008 Wiley-Liss, Inc. [source] Transplanted astrocytes internalize deposited ,-amyloid peptides in a transgenic mouse model of Alzheimer's diseaseGLIA, Issue 2 2008Rea Pihlaja Abstract Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. The neuropathological hallmarks include extracellular senile plaques consisting of deposited ,-amyloid (A,) peptides and intraneuronal neurofibrillary tangles. Neuroinflammation and activation of astrocytes are also well-established features of AD neuropathology; however, the relationships between astrocytes and A, deposition remain unclear. Previous studies have shown that adult mouse astrocytes internalize and degrade A, deposits in brain sections prepared from human amyloid precursor protein (APP) transgenic mice. In the present study, we demonstrate that cultured adult, but not neonatal mouse astrocytes, respond morphologically and degrade A, deposits present in human AD brain. We also transplanted astrocytes isolated from enhanced green fluorescent protein expressing adult and neonatal mice into the hippocampi of human A, plaque-bearing transgenic APPSwe+PS1dE9 (APdE9) mice and their wild-type littermates and followed the migration and localization of these astrocytes by confocal microscopy upto 7 days after transplantation. Posttransplantation the astrocytes localized as aggregates or thin strings of many cells within the hippocampi of APdE9 and wild-type mice and showed limited migration from the injection site. Interestingly, most of the transplanted astrocytes were found near A, deposits in the hippocampi of APdE9 mice. In contrast to findings in ex vivo degradation assay, confocal microscopy revealed that both adult and neonatal transplanted astrocytes internalized human A, immunoreactive material in vivo. These results support the role of astrocytes as active A, clearing cells in the CNS that may have important implications for future development of therapeutic strategies for AD. © 2007 Wiley-Liss, Inc. [source] DEC-205lo Langerinlo neonatal Langerhans' cells preferentially utilize a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigenIMMUNOLOGY, Issue 4 2003Bernadette M. Bellette Summary Antigen treatment of neonatal epidermis results in antigen-specific immune suppression. Compared with adult counterparts, neonatal Langerhans' cells (LC) demonstrate an impaired ability to transport antigen to the lymph node (LN). As it is possible that neonatal LC have a reduced ability to endocytose antigen, we evaluated the acquisition of endocytic function, the expression of uptake receptors and the internalization of soluble and small particulate antigens in neonatal, juvenile and adult mice. Although LC from 4-day-old mice were weakly positive for the mannose-type receptor, Langerin, they were capable of internalizing fluorescein isothiocyanate (FITC)-dextran, but to a lesser extent than LC from 6-week-old mice. However, when ratio data were calculated to account for variations in fluorescence intensity at 4°, it was demonstrated that neonatal LC continued to internalize antigen over a longer period of time than adult mice and, as the ratios were much higher, that neonatal cells were also relatively more efficient in antigen uptake. When receptors for mannan and mannose were competitively blocked, LC from neonatal mice, but not adult mice, could still efficiently internalize FITC,dextran. Consequently, the uptake of FITC,dextran, in part, occurred via alternative receptors or a receptor-independent fluid-phase pathway. A feasible pathway is macropinocytosis, as LC from 4-day-old mice demonstrated a reduction in FITC,dextran internalization by the macropinocytosis inhibitor, wortmannin. Evidence of a functional macropinocytosis pathway in neonatal LC was further supported by internalization of the soluble tracer Lucifer Yellow (LY). We conclude that neonatal LC preferentially utilize a wortmannin-sensitive, fluid-phase pathway, rather than receptor-mediated endocytosis, to internalize antigen. As neonatal LC are capable of sampling their environment without inducing immunity, this may serve to avoid inappropriate immune responses during the neonatal period. [source] Critical analysis of potential body temperature confounders on neurochemical endpoints caused by direct dosing and maternal separation in neonatal mice: a study of bioallethrin and deltamethrin interactions with temperature on brain muscarinic receptorsJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2003Jürgen Pauluhn Abstract The present investigation was conducted to understand better possible confounding factors caused by direct dosing of neonatal mice during the pre-weaning developmental period. By direct dosing, pups might encounter thermal challenges when temporarily removed from their ,natural habitat'. Typically, this leads to a cold environment and food deprivation (impaired lactation) and modulation of the toxic potency of the substance administered. Growth retardation as a consequence of such behavioural changes in pups makes it increasingly difficult to differentiate specific from non-specific mechanisms. Neonatal NMRI mice were dosed daily by gavage (0.7 mg kg,1 body wt.) from postnatal day (PND) 10,16 with S -bioallethrin, deltamethrin or the vehicle. Then the pups, including their non-treated foster dams, were subjected temporarily for 6 h day to a hypo-, normo- or hyperthermic environment, which was followed by normal housing. The measured temperatures in the environmental chambers were ca. 21, 25 and 30°C, respectively. Thus, temperatures in the hypo- and normothermic groups are comparable to the temperatures commonly present in testing laboratories, whereas the hyperthermic condition is that temperature typically present in the ,natural habitat' of pups. A deviation from the normal behaviour of both pups and dams was observed in the hypo- and normothermic groups. In these groups the rectal temperatures of pups were markedly decreased, especially in the early phase of the study (PND 10,12). Neonates that received either test substance displayed changes in body weights and brain weights at terminal sacrifice (PND 17) when subjected temporarily to a non-physiological environment. An enormous influence of environmental temperature on the density of muscarinic receptors in the crude synaptosomal fraction of the cerebral cortex was ascertained. In summary, these results demonstrate that the direct dosing of thermolabile neonatal mice by gavage is subject to significant artefacts that render the interpretation of findings from such studies difficult. It appears that if direct dosing of neonatal pups is mandated, and inhalation is a relevant route of exposure, the combined inhalation exposure of dams with their litters is an alternative procedure that does not cause disruption of the ,natural habitat' of pups. However, owing to their higher ventilation, under such conditions the pups may receive dosages at least double those of the dams. Copyright © 2003 John Wiley & Sons, Ltd. [source] Role of histamine in short- and long-term effects of methamphetamine on the developing mouse brainJOURNAL OF NEUROCHEMISTRY, Issue 4 2008Summer F. Acevedo Abstract With the rise in methamphetamine (MA) use among women of childbearing age, the potential consequences of MA exposure to the developing brain for cognition in adulthood is a major concern. Histamine might mediate these MA effects. Following MA administration in neonatal mice, histamine levels in brain were elevated and the hypothalamic-pituitary-adrenal axis was activated. Co-administration of MA with the H3 receptor agonist immepip antagonized these effects. The effects of MA on histamine levels and on hypothalamic-pituitary-adrenal axis activation at P20 were more pronounced in female than male mice. These sex differences could have contributed to the increased susceptibility of female mice to the detrimental long-term cognitive effects of MA and the H3/H4 antagonist thioperamide. Following behavioral testing, mice neonatally treated with MA or thioperamide showed reduced levels of the dendritic marker microtubule-associated protein 2 in the CA3 region of the hippocampus and the enthorhinal cortex. This was not seen in mice neonatally treated with immepip and MA who did not show cognitive impairments, suggesting that these brain areas might be particularly important for the long-term effects of MA on cognitive function. These data support a role for histamine in the effects of MA on the developing brain. [source] Pheomelanin Production in the Epidermis from Newborn Agouti Mice is Induced by the Expression of the Agouti Gene in the DermisPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2004Tomohisa Hirobe The present study was designed to clarify the role of the agouti gene in the regulation of the proliferation and differentiation of mouse epidermal melanocytes using serum-free primary culture of epidermal melanocytes from 0.5-d-old black (a/a; C57BL/10JHir) mice and congenic, agouti (A/A; C57BL/10JHir- A/A) mice. There was no significant difference in the proliferation or differentiation of melanocytes between a/a and A/A mice. However, the content of pheomelanin in culture media from A/A melanocytes was increased by l -tyrosine compared with a/a melanocytes. In addition, the content of the pheomelanin precursor, 5- S -cysteinyldopa, in culture media from A/A melanocytes was dramatically increased by l -tyrosine. Moreover, pheomelanin content in the epidermis from 3.5- and 5.5-d-old A/A mice was much higher than in a/a mice. Analysis of the A gene using reverse transcription-polymerase chain reaction revealed that cultured keratinocytes and melanocytes do not express the A gene. Moreover, the A gene was expressed in the A/A dermis of 0.5-, 3.5- and 5.5-d-old mice, but not in the a/a dermis nor in the A/A or a/a epidermis. These results suggest that A/A epidermal melanoblasts are influenced by the A gene from the dermis of neonatal mice, and are capable of synthesizing pheomelanin in the culture. Pheomelanin production in the epidermis from 3.5- and 5.5-d-old A/A mice may be induced by the expression of the agouti gene in the dermis. [source] Trafficking of macromolecules and organelles in cultured Dystonia musculorum sensory neurons is normalTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2006Madeline Pool Abstract Dystonia musculorum (dt) mice suffer from a recessive neuropathy characterized by the progressive loss of sensory axons. The gene responsible for this disorder, dystonin/Bpag1, encodes several alternatively spliced forms of a cytoskeletal linker protein. Neural isoforms of dystonin/Bpag1 are predicted to link actin filaments to microtubules. Consistent with this, previous observations have demonstrated that the cytoskeleton within sensory neurites of dt mice is perturbed. Also, recent results have indicated that a neural isoform of dystonin/Bpag1 interacts with the dynein motor complex. Because microtubule organization and dynein motor function are essential for trafficking, we hypothesized that this process would be perturbed in dt sensory neurons. Here, we demonstrate that cultured primary dorsal root ganglion (DRG) neurons express dystonin/Bpag1 and that loss of this expression causes an increase in apoptosis and a decrease in average neurite length. In contrast, detailed examination showed that the organization of microtubules is indistinguishable in DRG neuronal cultures from neonatal dt and wild-type mice. In addition, the steady-state distribution of several molecules and organelles is unchanged in these cultures. Furthermore, the speeds of mitochondrial movement in both anterograde and retrograde directions were comparable in dt and wild-type sensory neurons cultured from neonatal mice. Thus, dystonin/Bpag1 is not essential for microtubule network assembly since the microtubule network is intact in short-term cultures of sensory neurons from neonatal mice lacking this protein. In addition, dystonin/Bpag1 is not an essential part of the dynein motor complex for mitochondrial transport since mitochondrial trafficking is normal in cultured sensory neurons from dt mice. J. Comp. Neurol. 494:549,558, 2006. © 2005 Wiley-Liss, Inc. [source] Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal miceTHE JOURNAL OF GENE MEDICINE, Issue 4 2006Elena Fedorova Abstract Background Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal. Methods Four weeks after intrathecal injection into the spinal canal of newborn mice, the expression of the LacZ gene was assessed by histochemical staining and by in situ polymer chain reaction (PCR). Following the spinal infusion of a lentivector carrying the SEAP gene, levels of enzymatically active SEAP were measured in the CSF, blood serum, and in brain extracts. Results Intrathecal spinal canal delivery of lentivectors to newborn mice resulted in patchy, widely scattered areas of ,-gal expression mostly in the meninges. The transduction of the meningeal cells was confirmed by in situ PCR. Following the spinal infusion of a lentivector carrying the SEAP gene, sustained presence of the reporter protein was detected in the CSF, as well as in blood serum, and brain extracts. Conclusions These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier. Copyright © 2006 John Wiley & Sons, Ltd. [source] Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditionsARTHRITIS & RHEUMATISM, Issue 3 2010Atsushi Fukai Objective To examine the role of the phosphoinositide-dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification. Methods Skeletal phenotypes of homozygous Akt1-deficient (Akt1,/,) mice and their wild-type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1. Results Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1,/, chondrocytes, indicating a dominant role of Akt1. Akt1,/, mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild-type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1,/, joints. Calcification was significantly suppressed in cultures of Akt1,/, chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1. Conclusion Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA. [source] Regulation of autophagy in human and murine cartilage: Hypoxia-inducible factor 2 suppresses chondrocyte autophagyARTHRITIS & RHEUMATISM, Issue 5 2009Jolene Bohensky Objective We have previously demonstrated that the transcription factor hypoxia-inducible factor 1 (HIF-1) promotes the onset of autophagy in chondrocytes. The overall goal of this study was to test the hypothesis that another HIF family transcription factor, HIF-2, modulates the induction of autophagy by chondrocytes. Methods Expression of HIF-1, HIF-2, and light chain 3 (LC3) in human and murine articular cartilage was visualized by immunohistochemistry. Suppression of HIF-2 was achieved using small interfering RNA technology. Assessments of autophagic flux and lysosomal activity, as well as ultrastructural analysis, were performed in chondrocytes in cell culture. Results HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2, was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1 and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2,,knockout mouse growth plate showed an elevated autophagic response throughout the plate. Conclusion Based on these observations, we conclude that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Our data suggest that this protein acts as a brake on the autophagy-accelerator function of HIF-1. [source] Injections of Blood, Thrombin, and Plasminogen More Severely Damage Neonatal Mouse Brain Than Mature Mouse BrainBRAIN PATHOLOGY, Issue 4 2005Mengzhou Xue MD The mechanism of brain cell injury associated with intracerebral hemorrhage may be in part related to proteolytic enzymes in blood, some of which are also functional in the developing brain. We hypothesized that there would be an age-dependent brain response following intracerebral injection of blood, thrombin, and plasminogen. Mice at 3 ages (neonatal, 10-day-old, and young adult) received autologous blood (15, 25, and 50 ,l respectively), thrombin (3, 5, and 10 units respectively), plasminogen (0.03, 0.05, and 0.1 units respectively) (the doses expected in same volume blood), or saline injection into lateral striatum. Forty-eight hours later they were perfusion fixed. Hematoxylin and eosin, lectin histochemistry, Fluoro-Jade, and TUNEL staining were used to quantify changes related to the hemorrhagic lesion. Damage volume, dying neurons, neutrophils, and microglial reaction were significantly greater following injections of blood, plasminogen, and thrombin compared to saline in all three ages of mice. Plasminogen and thrombin associated brain damage was greatest in neonatal mice and, in that group unlike the other 2, greater than the damage caused by whole blood. These results suggest that the neonatal brain is relatively more sensitive to proteolytic plasma enzymes than the mature brain. [source] Contrasting effects of basic fibroblast growth factor and epidermal growth factor on mouse neonatal olfactory mucosa cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Perrine Barraud Abstract Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) affect proliferation and survival of many cell types, but their role in the maintenance of olfactory mucosa cells remains unclear. In the neonatal mouse olfactory mucosa, cell proliferation mainly occurs in the neuroepithelium and, to a lesser extent, in the lamina propria. To establish whether bFGF and EGF affect proliferation and/or survival of these cells, we isolated olfactory mucosa cells from the neonatal mouse and cultured them as free-floating spheres under bFGF or EGF stimulation. Our data demonstrate that bFGF is a mitogen for the rapidly dividing cells (olfactory neuronal precursors and olfactory ensheathing cells), and also a survival factor for both slowly and rapidly dividing cells of the olfactory mucosa. In contrast, EGF appears to be primarily a survival factor for both the olfactory stem and precursor cells. [source] Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal miceGLIA, Issue 8 2009Kai Härtel Abstract A controlled, periodic exchange of air between lungs and atmosphere requires a neuronal rhythm generated by a network of neurons in the ventral respiratory group (VRG) of the brainstem. Glial cells, e.g. astrocytes, have been shown to be supportive in stabilizing this neuronal activity in the central nervous system during development. In addition, a variety of neuromodulators including serotonin (5-HT), Substance P (SP), and thyrotropin-releasing hormone (TRH) stimulate respiratory neurons directly. If astrocytes in the VRG, like their neuronal neighbors, are also directly stimulated by neuromodulators, they might indirectly affect the respiratory neurons and consequently the respiratory rhythm. In the present study, we provide support for this concept by demonstrating expression of NK1-R, TRH-R, and 5-HT2 -R in astrocytes of the VRG with immunohistochemistry. Additionally, we showed that the external application of the neuromodulators 5-HT, SP, and TRH activate calcium transients in VRG astrocytes. Consequently, we postulate that in the VRG of the neonatal mouse, neuromodulation by SP, TRH, and serotonin also involves astrocytic calcium signaling. © 2008 Wiley-Liss, Inc. [source] Endogenous extracellular serotonin modulates the spinal locomotor network of the neonatal mouseTHE JOURNAL OF PHYSIOLOGY, Issue 1 2010Mary J. Dunbar Serotonin (5-HT) can potently activate and modulate spinal locomotor circuits in a variety of species. Many of these findings have been obtained by applying serotonin exogenously to the isolated spinal cord of in vitro preparations, which has the drawback of indiscriminately activating extrasynaptic receptors and neurons. To investigate the role of endogenously released serotonin in modulating locomotor networks, the selective serotonin reuptake inhibitor citalopram was used. Fictive locomotion was elicited by either electrical stimulation of the brainstem or the sacral 4 (S4) dorsal root. The addition of 20 ,m of citalopram caudal to thoracic segment 5 (T5) had an overall inhibitory effect on the lumbar central pattern generator (CPG). Left,right and flexor,extensor coupling were significantly decreased, and there was also a phase shift in the flexor,extensor relationship. In addition, there was a significant decrease in burst amplitude. These effects were observed during both afferent and brainstem evoked fictive locomotion. When citalopram was added in the presence of 5-HT1A and 5-HT1B antagonists, the inhibitory effects were largely reversed. The remaining excitatory effects were mediated by 5-HT7 and 5-HT2 receptors. These results suggest that endogenous 5-HT release can modulate locomotor-like activity early in neonatal development. [source] | |||||||||||||