Newborn Mouse Pups Research Articles

Postnatal Undernutrition (PUN) Permanently Alters the Heart to Reduce Maximal Exercise Capacity in Female Mice

Undernutrition during development increases the risk for adult cardiovascular disease. To determine if PUN alters heart structure function, and maximal exercise capacity in adulthood, newborn control mouse pups were fostered to dams fed a control (CON; 20% protein) or a protein-restricted diet (PUN; 8% protein; n=7-10 litters/treatment). At 21 d (PN21) pups were fed the CON diet ad lib. At PN70 a maximal treadmill test was performed to determine VO2peak; maximal vertical work was calculated for the final 30 sec. At PN80 cardiac function was evaluated by echocardiography. VO2peak was the same in CON and PUN mice and higher in males than females (249 + 9 and 169 + 6 ml/hr, respectively, P<0.001); gender x diet, NS. Maximal vertical work at VO2peak was higher in CON than PUN females (1.64 + 0.07 and 1.33 + 0.06 J/min, respectively, P < 0.01) but not males; gender x diet, P<0.02. Resting cardiac output was higher in CON than PUN offspring (18.8 + 0.7 and 16.3 + 0.7 ml/min, respectively; P<0.02) and in males than females (19.2 + 0.7 and 15.9 + 0.7 ml/min, respectively; P<0.01; gender x diet, NS) due to differences in stroke volume (CON: 43.0 ± 1.5; PUN: 37.7 ± 1.4 µl; P<0.05; males: 46.8 ± 1.5; females: 34.0 ± 1.4 µl; P<0.05), and left ventricular mass (CON: 104 ± 5; PUN: 90 ± 5 mg; P<0.05; males: 108 ± 5; females: 90 ± 5 mg; P<0.05). Thus, PUN permanently reduces left ventricular mass which in turn reduces stroke volume and maximal treadmill work capacity in female mice. Male mice were able to compensate for the reduction in cardiac output and maintain maximal exercise capacity. Supported by NIH AR46308, T32HL07676, USDA/ARS 6250-51000-051

Spatio-temporal regulation of the formation of the somatosensory system.

The somatosensory system in the brain has been widely used for investigating the mechanisms underlying neural circuit formation and developmental neural plasticity. In the primary somatosensory cortex (S1) of rodents, there are discrete cytoarchitectonic units called barrels. Reverse genetic analyses using knockout mice have revealed molecules that control spatial pattern formation of barrels in S1. Glutamatergic receptors such as the NMDA receptor and mGluR5, and molecules related to serotonin such as serotonin transporter and monoamine oxidase A are essential for the formation of barrels. In addition to the mechanisms of spatial pattern formation, those regulating the timing of developmental processes were uncovered recently. Barrels are formed soon after the birth of newborn mouse pups from their mothers, and it was shown that the timing of barrel formation was determined by the timing of the birth of mouse pups. The mechanisms downstream of birth were also examined. It would be intriguing to examine if the mechanisms found using the somatosensory system are applicable to other brain regions.

Activin A contributes to the development of hyperoxia-induced lung injury in neonatal mice.

Bronchopulmonary dysplasia (BPD) is one of the leading causes of morbidity and mortality in babies born prematurely, yet there is no curative treatment. In recent years, a number of inhibitors against TGFβ signaling have been tested for their potential to prevent neonatal injury associated with hyperoxia, which is a contributing factor of BPD. In this study, we assessed the contribution of activin A-a member of the TGFβ superfamily-to the development of hyperoxia-induced lung injury in neonatal mice. We placed newborn C57Bl6 mouse pups in continuous hyperoxia (85% O2) to mimic many aspects of BPD including alveolar simplification and pulmonary inflammation. The pups were administered activin A receptor type IIB-Fc antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on postnatal days 4, 7, 10, and 13. Treatment with ActRIIB-Fc and follistatin protected against hyperoxia-induced growth retardation. ActRIIB-Fc also reduced pulmonary leukocyte infiltration, normalized tissue: airspace ratio and increased septal crest density. These findings were associated with reduced phosphorylation of Smad3 and decreased matrix metalloproteinase (MMP)-9 activity. This study suggests that activin A signaling may contribute to the pathology of bronchopulmonary dysplasia.

The Role of the Active Oxygen Produced from Gp91phox NADPH Oxidase on the Newborn Weight of Mouse Pups

It is known that active oxygen plays an important role in a reproduction. However, no report has so far investigated the influence of active oxygen produced from gp91phox NADPH oxidase on newborns. In this study, we investigated the influence of active oxygen on the weight of newborns using graviditas gp91phox-knockout (gp91phox-/-) mice. Gestational C57BL/6j (control), gp91phox-/-, and insulin-like growth factor-1-knockout (IGF-1-/-) mice were examined and the weight of the newborn mouse pups were analyzed. Gp91phox-/- and IGF-1-/- mouse pups had low weight compared with control mice. When the control mice were treated with an inhibitor of reactive oxygen species (ROS), the newborn weight decreased. Conversely, when the gp91phox-/- mice were treated with an activator of ROS, the newborn weight increased, however, it remained low in the IGF-1-/- mice. Moreover, there were decreased levels of IL-1 in the plasma of graviditas gp91phox-/- mice compared with control and IGF-1-/- mice. Treatment with an IL-1 receptor antagonist in the control mice resulted in a low newborn weight, similar to the gp91phox-/- and IGF-1-/- mice. Furthermore, the expression of NLRP3 and caspase-1 in the uterus of graviditas gp91phox-/- mice was low compared with the control and IGF-1-/- mice. These results clearly indicate that gp91phox NADPH oxidase produces ROS during graviditas. The ROS activate NLRP3, and NLRP3 leads to the production of caspase-1, which subsequently increases IL-1, thereby finally inducing IGF-1. Because the newborn weight is determined by IGF-1, gp91phox appears to be important for promoting fetal growth during graviditas

The response of newly born mice to odors of murine colostrum and milk: unconditionally attractive, conditionally discriminated.

It is a general rule that milk conveys chemosensory cues that are attractive to mammalian neonates. This study investigated whether compositional fluctuations in milk along lactation induce variations in newborn mouse pups' (Mus musculus, strain BALB/c) attraction to milk odor. Pups differing in suckling experience were exposed to the odor of milk sampled from females varying in lactational stage. Immediately after birth, suckling-inexperienced (P0) and suckling-experienced (P0suck ) pups were assayed in a series of paired-choice tests contrasting murine milk [of lactation days 0, 3, 15 (abridged L0, L3, L15, respectively)] and a blank (water) to evaluate olfactory detection and attraction of milk odor. Preference tests further paired these milk two-by-two to assess their relative attraction. Results showed first that P0 and P0suck pups detect and positively orient to any milk odor. When L0 is presented against L15 milk, P0 pups orient for a similar duration towards these odor stimuli, whereas P0suck pups spend more time toward the odor of L0 than of L15 milk. Finally, P0suck pups orient similarly to odors of L0 milk collected before/after the first suckling episode (L0 and L0suck , respectively), but the odor of L0 milk was more attractive than that of L3 milk. Thus, mouse pups' positive orientation toward the odors of murine colostrum (assumed to correspond to L0/L0suck milk) and later-lactation milk appears unconditional of previous suckling experience, whereas their ability to discriminate or display preference between milk differing in lactation stage appears conditional on postnatal exposure effects.

Maternal Dietary Docosahexaenoic Acid Supplementation Attenuates Fetal Growth Restriction and Enhances Pulmonary Function in a Newborn Mouse Model of Perinatal Inflammation1–3

The preterm infant is often exposed to maternal and neonatal inflammatory stimuli and is born with immature lungs, resulting in a need for oxygen therapy. Nutritional intervention with docosahexaenoic acid (DHA; 6.3 g/kg of diet) has been shown to attenuate inflammation in various human diseases. Previous studies demonstrated that maternal DHA supplementation during late gestation and lactation attenuated hyperoxic lung injury in newborn mouse pups. In the present studies, we tested the hypothesis that DHA supplementation to the dam would reduce hyperoxic lung injury and growth deficits in a more severe model of systemic maternal inflammation, including lipopolysaccharide (LPS) and neonatal hyperoxia exposure. On embryonic day 16, dams were placed on DHA (6.3 g DHA/kg diet) or control diets and injected with saline or LPS. Diets were maintained through weaning. At birth, pups were placed in room air or hyperoxia for 14 d. Improvements in birth weight (P < 0.01), alveolarization (P ≤ 0.01), and pulmonary function (P ≤ 0.03) at 2 and 8 wk of age were observed in pups exposed to perinatal inflammation and born to DHA-supplemented dams compared with control diet–exposed pups. These improvements were associated with decreases in tissue macrophage numbers (P < 0.01), monocyte chemoattractant protein-1 expression (P ≤ 0.05), and decreases in soluble receptor for advanced glycation end products concentrations (P < 0.01) at 2 and 8 wk. Furthermore, DHA supplementation attenuated pulmonary fibrosis, which was associated with the reduction of matrix metalloproteinases 2, 3, and 8 (P ≤ 0.03) and collagen mRNA (P ≤ 0.05), and decreased collagen (P < 0.01) and vimentin (P ≤ 0.03) protein concentrations. In a model of severe inflammation, maternal DHA supplementation lessened inflammation and improved lung growth in the offspring. Maternal supplementation with DHA may be a therapeutic strategy to reduce neonatal inflammation.

Routine clonal expansion of mesenchymal stem cells derived from amniotic fluid for perinatal applications

Stem cells (SCs) isolated from amniotic fluid (AF) are a promising source for autologous perinatal cell therapy. The aim of this study was to develop a routine isolation, selection, and expansion protocol of clonal SC lines from redundant clinical amniocentesis samples. Amniotic fluids were collected between 15 and 22 weeks of gestation, and SCs were isolated by CD117-based and mechanical selection protocols. SCs were characterized by mesenchymal SC marker expression and differentiation protocols. Cells were manipulated with a lentiviral vector system expressing the β-galactosidase reporter gene and were injected into immunodeficient newborn mouse pups. Qualitative assessment was performed to detect the infused cells after 1 week. A total of 78 clonal AF SC populations were successfully isolated by mechanical selection from 21 consecutive amniocentesis samples. They were positive for mesenchymal SC cluster of differentiation markers and could be differentiated into the different lineages. SCs were stably labeled using β-galactosidase and were detected in the lungs and hearts of the neonatal mice. We demonstrate that mesenchymal SCs can be routinely isolated and clonally expanded from mid-gestation human AF using mechanical isolation. They can easily be transduced and be tested for perinatal treatment in animal models.

High efficiency gene transfer to airways of mice using influenza hemagglutinin pseudotyped lentiviral vectors

A limitation to efficient lentivirus-mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. We investigated influenza hemagglutinin (HA) pseudotyped equine infectious anemia virus-derived lentiviral vector-mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies, high-titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re-dosing to the nasal airway. Delivery of high-titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in the efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose, vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung, approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re-dosing to the nasal airway of mice was found to be dependent of the age of the animal when the first dose is administered, as well as the length of time between doses. A single intranasal dose of concentrated influenza HA-pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases.

Identification of Label-Retaining Perivascular Cells in a Mouse Model of Endometrial Decidualization, Breakdown, and Repair1

The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a woman's reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.

Effects of Ionizing Radiation on Neural Precursor Cells (P02.023)

Objective: To elucidate cellular and molecular changes associated with the exposure of neural stem cells (NSCs) to high and low doses of ionizing radiation. Background Damage to normal human brain cells from targeted exposure to ionizing radiation may occur during the course of cancer radiotherapy or from accidental exposure. Delayed effects may complicate the acute immediate effects, and in particular have been associated with neurodegeneration and cognitive decline. These delayed effects may arise from the slow death of irradiated cells, damage to cells that survive the radiation exposure, or from negative effects on normal cell replenishment of non-targeted brain cells from neural stem cells. Design/Methods: Subventricular zone (SVZ) neural precursors isolated from newborn mouse pups were analyzed for survival, proliferation and differentiation after exposure to low and high doses of γ rays (range from 0 to 8 Gy). Results: The ability of the neurospheres to differentiate into astroglia, neurons, and oligodendroglia was assessed in the studied groups 8 days after irradiation. There were no significant changes in the neurosphere differentiation ability between the irradiated cells and controls (p>0.05). Surprisingly, the number of neurospheres formed showed no significant change from control, up to 8 Gy of γ irradiation. Meanwhile, the size of the spheres increased slightly at low doses without any significant change(0.4 or 0.5 Gy), and decreased dramatically at 8 Gy (p Conclusions: Our data suggest that neural stem cells/neural precursors derived from the SVZ are resistant to ionizing irradiation. However, their proliferation is inhibited subsequent to exposure to ionizing radiation. Supported by: Start up fund. Disclosure: Dr. Chen has nothing to disclose. Dr. Levison has nothing to disclose. Dr. De Toledo has nothing to disclose. Dr. Azzam has nothing to disclose. Dr. Souayah has received personal compensation for activities with Walgreens as a consultant. Dr. Souayah has received research support from Talecris.

Effects of Ionizing Radiation on Neural Precursor Cells (IN8-1.008)

Objective: To elucidate cellular and molecular changes associated with the exposure of neural stem cells (NSCs) to high and low doses of ionizing radiation. Background Damage to normal human brain cells from targeted exposure to ionizing radiation may occur during the course of cancer radiotherapy or from accidental exposure. Delayed effects may complicate the acute immediate effects, and in particular have been associated with neurodegeneration and cognitive decline. These delayed effects may arise from the slow death of irradiated cells, damage to cells that survive the radiation exposure, or from negative effects on normal cell replenishment of non-targeted brain cells from neural stem cells. Design/Methods: Subventricular zone (SVZ) neural precursors isolated from newborn mouse pups were analyzed for survival, proliferation and differentiation after exposure to low and high doses of γ rays (range from 0 to 8 Gy). Results: The ability of the neurospheres to differentiate into astroglia, neurons, and oligodendroglia was assessed in the studied groups 8 days after irradiation. There were no significant changes in the neurosphere differentiation ability between the irradiated cells and controls (p>0.05). Surprisingly, the number of neurospheres formed showed no significant change from control, up to 8 Gy of γ irradiation. Meanwhile, the size of the spheres increased slightly at low doses without any significant change(0.4 or 0.5 Gy), and decreased dramatically at 8 Gy (p Conclusions: Our data suggest that neural stem cells/neural precursors derived from the SVZ are resistant to ionizing irradiation. However, their proliferation is inhibited subsequent to exposure to ionizing radiation. Supported by: Start up fund. Disclosure: Dr. Chen has nothing to disclose. Dr. Levison has nothing to disclose. Dr. De Toledo has nothing to disclose. Dr. Azzam has nothing to disclose. Dr. Souayah has received personal compensation for activities with Walgreens as a consultant. Dr. Souayah has received research support from Talecris.

Effects of Oxidative Stress on Intestinal Type I Insulin-Like Growth Factor Receptor Expression

Oxidative stress activates multiple signaling transduction pathways, including the phosphatidylinositol 3-kinase (PI3-K), in an injured intestine as occurs in necrotizing enterocolitis (NEC). We have previously shown that hydrogen peroxide (H2O2)-induced PI3-K activation is significantly enhanced with exogenous insulin-like growth factor (IGF)-1 in intestinal epithelial cells. However, the effects of oxidative stress on IGF receptor type I (IGF-IR) activation and expression in the neonatal intestine during NEC are unknown. Intestinal sections from neonates undergoing bowel resections (control = 3, NEC = 20) were analyzed for IGF-IR expression. NEC was induced in newborn mouse pups using hypoxia and hyperosmolar feeds, and distal small bowel segments were analyzed for IGF-IR expression (control = 3, NEC = 7). H2O2 was used to induce oxidative stress in rat (RIE-1) and fetal human (FHs74 Int) intestinal epithelial cells. Phosphorylation of IGF-IR, Akt, a downstream effector of PI3-K, and IGF-IR levels were determined by Western blotting. Flow cytometry, immunofluorescence, immunohistochemistry, IGF-IR tyrosine phosphorylation array, cell death enzyme-linked immunosorbent assay, and Western blotting were used to determine the IGF-IR expression. An increased IGF-IR expression was noted in intestinal sections from NEC as well as murine model of NEC. H2O2 treatment rapidly activated IGF-IR and increased the expression in RIE-1 and FHs74 Int cells. Inhibition of IGF-IR resulted in significant RIE-1 cell apoptosis during oxidative stress. IGF-IR tyrosine phosphorylation array showed the recruitment of several key SH2 domain-containing proteins and oncogenes to the IGF-IR tyrosine kinase domain in H2O2-treated RIE-1 cells. IGF-IR-mediated activation of intracellular signaling may play a critical role during oxidative stress-induced apoptosis in NEC.

Maternal Docosahexaenoic Acid Supplementation Decreases Lung Inflammation in Hyperoxia-Exposed Newborn Mice1,2

DHA is a long-chain fatty acid that has potent antiinflammatory properties. Whereas maternal DHA dietary supplementation has been shown to improve cognitive development in infants fed DHA-supplemented milk, the antiinflammatory effects of maternal DHA supplementation on the developing fetus and neonate have not been extensively explored. Pregnant C3H/HeN dams were fed purified control or DHA-supplemented diets (~0.25% of total fat) at embryonic d 16 and consumed these diets throughout the study. At birth, the nursing mouse pups were placed in room air (RA; 21% O(2)) or >95% O(2) (hyperoxia) for up to 7 d. These studies tested the hypothesis that maternal DHA supplementation would decrease inflammation and improve alveolarization in the lungs of newborn mouse pups exposed to hyperoxia. Survival, inflammatory responses, and lung growth were compared among control diet/RA, DHA/RA, control/O(2), and DHA/O(2) pups. There were fewer neutrophils and macrophages in lung tissues from pups nursed by DHA-supplemented dams than in those nursed by dams fed the control diet at 7 d of hyperoxia exposure (P < 0.015). Although differences due to hyperoxia exposure were observed, maternal diet did not affect keratinocyte-derived chemokine, macrophage inflammatory protein-2, IL-1β, or TNFα mRNA levels in pup tissues. Hyperoxia also induced NF-κB activity, but maternal diet did not affect NF-κB or PPARγ activities. In mice, DHA supplementation decreases leukocyte infiltration in the offspring exposed to hyperoxia, suggesting a potential role for DHA supplementation as a therapy to reduce inflammation in preterm infants.

Prevention of the murine model of biliary atresia after live rotavirus vaccination of dams

Purpose Biliary atresia (BA) is a neonatal disease that results in the obliteration of the biliary tree. The murine model of BA has been established where rhesus rotavirus (RRV) infection of newborn mice leads to an obstructive cholangiopathy. We determined whether maternal postconception rotavirus vaccination could prevent the murine model of BA. Materials and Methods Female mice were mated and injected intraperitoneally with one of the following materials: purified rotavirus strains RRV or Wa, high or low-dose Rotateq (Merck and Co Inc, Whitehouse Station, NJ) (a pentavalent rotavirus vaccine [PRV]), purified recombinant viral antigens of rotavirus (VP6) or influenza (NP), or saline. B-cell–deficient females also underwent postconception PRV injection. Results Maternal vaccination with PRV improves survival of pups infected with RRV. Serum rotavirus IgG, but not IgA, levels were increased in pups delivered from dams who received RRV, Wa, PRV, or VP6, but in the case of the Wa, PRV, and VP6 groups, these antibodies were not neutralizing. Postconception injection of high-dose PRV did not improve survival of pups born to B-cell–deficient dams. Conclusion Maternal vaccination against RRV can prevent the rotavirus-induced murine model of BA in newborn mouse pups.

Differential Responses in the Lungs of Newborn Mouse Pups Exposed to 85% or &gt;95% Oxygen

Premature infants often develop serious clinical complications associated with respiratory failure and hyperoxic lung injury that includes lung inflammation and alterations in lung development. The goal of these studies is to test the hypothesis that there are differences in the course of lung injury in newborn mice exposed to 85% or >95% oxygen that provide models to address the differential effects of oxidation and inflammation. Our results indicate differences between the 85% and >95% O2 exposure groups by day 14 in weight gain and lung alveolarization. Inflammation, assessed by neutrophil counts, was observed in both hyperoxia groups by day 3 but was dramatically greater in the >95% O2-exposed groups by day 14 and associated with greater developmental deficits. Cytoplasmic phospholipase A2, cyclooxygenase-2, and 5-lipoxygenase levels were elevated but no patterns of differences were observed between exposure groups. Prostaglandins D2, E2, and F2alpha were increased in the tissues from mouse pups exposed to >95% O2 at 7 d indicating a differential expression of cyclooxygenase-2 products. Our data indicate that there are differences in the models of 85% or >95% O2 exposure and these differences may provide mechanistic insights into hyperoxic lung injury in an immature system.

N- and C-terminal cooperation in rotavirus enterotoxin: novel mechanism of modulation of the properties of a multifunctional protein by a structurally and functionally overlapping conformational domain.

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic alpha-helix located between aa 73 and 85 (AAH73-85) at the N terminus of DeltaN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.

Adeno-associated virus type-2 expression of pigmented epithelium-derived factor or Kringles 1-3 of angiostatin reduce retinal neovascularization.

Neovascular diseases of the retina include age-related macular degeneration and diabetic retinopathy, and together they comprise the leading causes of adult-onset blindness in developed countries. Current surgical, pharmaceutical, and laser therapies for age-related macular degeneration (AMD) rarely result in improved vision, do not significantly prevent neovascularization (NV), and often result in at least some vision loss. To address this therapeutic gap, we determined the efficacy of recombinant adeno-associated viral (rAAV) serotype-2-mediated expression of pigment epithelium-derived factor (PEDF) or Kringle domains 1-3 of angiostatin (K1K3) in reducing aberrant vessel formation in a mouse model of ischemia-induced retinal NV. Both PEDF and K1K3 are potent inhibitors of NV when injected directly, hence expression of these therapeutic factors from rAAV may provide long-term protection from neovascular eye disease. rAAV vectors expressing the therapeutic gene were injected into one eye of postnatal day 0 (P0) newborn mouse pups. Retinal NV was induced in P7 mice by exposure to elevated oxygen for 5 days followed by room air for another five days. Retinal NV was quantified by the number of vascular-endothelial-cell nuclei above the inner-limiting membrane in P17 eyes. The number of such vascular endothelial cell nuclei in eyes treated with rAAV-PEDF or rAAV-K1K3 was significantly reduced (both P < 0.0000002) compared with control eyes. Ocular protein levels detected by ELISA correlate well with the reduction in NV and confirm that expression of antineovascular agents from rAAV vectors may be a therapeutically useful treatment of retinal or choroidal neovascular disease.

A rapid and reliable PCR-based assay for gene transmission and sex determination in newborn transgenic mice.

This article describes a reliable and rapid method for simultaneous detection of a transgene and sex determination in the newborn mouse pups by PCR using three sets of primers in a single reaction. One set of sense/antisense primers is used to amplify the experimental transgene (androgen receptor gene in this case), the second set for the mouse Y-chromosome-specific SRY gene, and the third set for the beta subunit of the thyroid stimulating hormone (TSH beta), an internal control. This procedure allowed us to promptly analyze pups born from transgenic founders carrying the androgen receptor transgene and, at the same time, establish the sex of the animals. The method is simple, rapid and highly reproducible.

Characterization of two distinct opsonic and protective epitopes within the alpha C protein of the group B Streptococcus.

Group B Streptococcus (GBS) is a major cause of neonatal sepsis, meningitis in early infancy, postpartum endometritis, and serious invasive infections in adults in the United States. We previously cloned, sequenced, and characterized the alpha antigen gene, bca, and showed that the alpha C protein of GBS is a trypsin-resistant, surface-associated polypeptide that contains a signal sequence, a unique N terminus, nine identical tandem repeats, and a C-terminal membrane anchor structure. Polyclonal antiserum raised to the recombinant alpha C protein and an opsonic monoclonal antibody, 4G8, raised to the native protein from GBS have been shown to be protective in a mouse model. The binding site of 4G8 has now been localized to the tandem repeat region of the alpha C protein. To determine whether the N terminus of the alpha C protein contains additional opsonic and/or protective epitopes, the sequence corresponding to the alpha C protein N terminus was subcloned into a pET vector, the expressed peptide from Escherichia coli was purified by Ni2+ affinity chromatography, and rabbit polyclonal antibodies were raised to the purified recombinant peptide. Antibodies to the alpha C protein N terminus were shown to be opsonic by an in vitro opsonophagocytosis assay. In addition, 69% of newborn mouse pups from mothers passively immunized with the antiserum to the recombinant N-terminal polypeptide of the alpha C protein were protected against lethal challenge with GBS A909. These data indicate that at least two distinct regions of the alpha C protein, the N terminus and the tandem repeat region, contain opsonic and protective epitopes.

Fine structure of dividing astroglia and oligodendroglia during myelin formation in the developing mouse spinal cord.

To study the morphology and cellular relationships of dividing glial cells during myelin formation, were perfused newborn and 5-day mouse pups and embedded slices of cervical, thoracic, and lumbar cord for light and electron microscopic study. In semithin epon sections stained with toluidine blue, all levels of spinal cord at both ages contained mitotic glia in gray columns and funiculi. In electron micrographs of funiculi, dividing astroglia containing bundles of glial filaments, many glycogen granules, and had large processes extending into the surrounding neuropil. Cytoplasmic organelles of many immature interphase oligodendroglia and mitotic oligodendroblasts were similar and included microtubules, clusters of free ribosomes, and scattered profiles of granular endoplasmic reticulum. Unlike astroglia, dividing oligodendroblasts lacked large processes and in metaphase they were ellipsoids and had smooth plasma membranes. When these cells were studied in alternating serial thin and semithin sections over 10-15 micrometers distances, we did not identify connections between myelin sheaths and mitotic oligodendroblasts. Our findings indicate that oligodendroglia in developing white matter multiply before developing large processes. Our data also suggest that oligodendroglia do not divide while forming myelin.