LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1(-/-)) on a low-density lipoprotein receptor-deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. APPROACH AND RESULTS: We found smLRP1(-/-) mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1(-/-) vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. CONCLUSIONS: Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

beta1 integrin maintains integrity of the embryonic neocortical stem cell niche.

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin alpha2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.

Glucocorticoid exposure of sheep at 0.7 to 0.75 gestation augments late-gestation fetal stress responses.

OBJECTIVES: Exposure to glucocorticoid levels inappropriately high for current maturation alters fetal hypothalamo-pituitary-adrenal axis (HPAA) development. In an established fetal sheep model, we determined whether clinical betamethasone doses used to accelerate fetal lung maturation have persistent effects on fetal HPAA hypotensive-stress responses. STUDY DESIGN: Pregnant ewes received saline (n = 6) or betamethasone (n = 6); 2 × 110 µg/kg body weight doses injected 24 hours apart (106/107 and 112/113 days' gestational age, term 150 days). Basal adrenocorticotropin (ACTH) and cortisol and responses to fetal hypotension were measured before and 5 days after the first course and 14 days after the second course. RESULTS: Basal ACTH and cortisol were similar with treatment. HPAA responses to hypotension increased after the second but not first course and ACTH/cortisol ratio increased indicating central HPAA effects. CONCLUSIONS: Results demonstrate latency in the emergence of fetal HPAA hyperresponsiveness following betamethasone exposure that may explain hyperresponsiveness in full-term but not preterm neonates.

Air-Evacuation-Relevant Hypobaria Following Traumatic Brain Injury Plus Hemorrhagic Shock in Rats Increases Mortality and Injury to the Gut, Lungs, and Kidneys.

ABSTRACT: Rats exposed to hypobaria equivalent to what occurs during aeromedical evacuation within a few days after isolated traumatic brain injury exhibit greater neurologic injury than those remaining at sea level. Moreover, administration of excessive supplemental O2 during hypobaria further exacerbates brain injury. This study tested the hypothesis that exposure of rats to hypobaria following controlled cortical impact (CCI)-induced brain injury plus mild hemorrhagic shock worsens multiple organ inflammation and associated mortality. In this study, at 24 h after CCI plus hemorrhagic shock, rats were exposed to either normobaria (sea level) or hypobaria (=8,000 ft altitude) for 6 h under normoxic or hyperoxic conditions. Injured rats exhibited mortality ranging from 30% for those maintained under normobaria and normoxia to 60% for those exposed to 6 h under hypobaric and hyperoxia. Lung histopathology and neutrophil infiltration at 2 days postinjury were exacerbated by hypobaria and hyperoxia. Gut and kidney inflammation at 30 days postinjury were also worsened by hypobaric hyperoxia. In conclusion, exposure of rats after brain injury and hemorrhagic shock to hypobaria or hyperoxia results in increased mortality. Based on gut, lung, and kidney histopathology at 2 to 30 days postinjury, increased mortality is consistent with multi-organ inflammation. These findings support epidemiological studies indicating that increasing aircraft cabin pressures to 4,000 ft altitude (compared with standard 8,000 ft) and limiting excessive oxygen administration will decrease critical complications during and following aeromedical transport.

Adverse effects of antenatal glucocorticoids on cerebral myelination in sheep.

OBJECTIVE: To determine in fetal sheep the effect of betamethasone on myelination in relation to stage of myelination, number of treatment courses, dose, and route of administration. METHODS: Fetal expression of myelin basic protein (MBP), a marker of mature oligodendrocytes and myelin, was determined between 0.27 and 0.93 gestation. Short-term betamethasone effects were examined 24 hours after one maternal intramuscular treatment course (weight adjusted to equal the clinical dose of 2 x 8 mg betamethasone to a 70-kg woman) at 0.63, 0.75, and 0.87 gestation or after continuous 48-hour fetal intravenous infusion at 0.75 and 0.87 gestation. Lasting effects were examined 20 days after one and two treatment courses weight-adapted to the clinical dose of 2 x 8 mg or 2 x 12 mg betamethasone at 0.75 gestation. RESULTS: Myelin basic protein immunoreactivity was first detected in the internal capsule at 0.53 gestation, followed by the centrum semiovale, the superficial white matter, and corpus callosum at 0.63 gestation. Within 24 hours after treatment, betamethasone reduced the number of mature oligodendrocytes and MBP immunoreactivity. The effect decreased with gestational age. Maternal and fetal betamethasone administration had similar effects. Loss of MBP immunoreactivity was not reversed 20 days after two treatment courses, independent of dose. CONCLUSION: Betamethasone-induced delayed cerebral myelination is dependent on the stage of brain development in sheep. Betamethasone-related disturbances in myelination and any potential contribution to childhood behavior deficits need to be confirmed in clinical studies.

Viable cryopreserved umbilical tissue (vCUT) reduces post-operative adhesions in a rabbit abdominal adhesion model.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) "point of care" allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at -80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.

Effects of antenatal glucocorticoids on cerebral substrate metabolism in the preterm ovine fetus.

OBJECTIVE: Although the benefits of antenatal glucocorticoids are well known for infants who are born preterm, there is increasing evidence of adverse effects on brain development, which may relate to altered metabolic activity. We have determined the effect of maternal glucocorticoid administration at doses that are used clinically on cerebral substrate metabolism in the preterm ovine fetus. STUDY DESIGN: Chronically instrumented pregnant sheep at 0.85 gestation received 2 intramuscular injections of betamethasone at 170 microg/kg maternal weight (n = 13) or saline (n = 10) 24 hours apart together with a continuous infusion of L-[1-(13)C] leucine to the fetus. Fetal cerebral substrate arteriovenous differences (O2, glucose, leucine, leucine enrichment) and blood flow (fluorescent microspheres) were measured at baseline, 24 hours after the first betamethasone/saline injection (late beta/saline 1), and 4 hours after the second betamethasone/saline injection (early beta/saline 2) to obtain substrate deliveries and fractional extractions. RESULTS: Fetal pH, blood gases, and metabolites were little changed in either group over the course of the study, except for glucose values in the betamethasone animals, which increased 1.4- and 1.9-fold, measured late beta 1 and early beta 2, respectively (both P < .01). Cerebral blood flow, although little changed in the control group or at late beta 1, was decreased at early beta 2 by approximately 30% (P < .05). As such, early beta 2 animals showed a decrease in cerebral O2 delivery of approximately 20% (P = .06) and conversely an increase in cerebral glucose delivery of 1.4- and 1.3-fold at late beta 1 (P < .05) and early beta 2 (P = .08), respectively. Fractional extraction values for these substrates were not changed significantly, which resulted in corresponding decreases in estimated O2 uptake and increases in estimated glucose uptake, such that the glucose/oxygen quotient (as an index of glucose oxidative metabolism) measured 1.6 at early beta 2, which was considerably greater than baseline values at 1.1 (P < .05). Fractional extraction values for leucine and leucine enrichment averaged 2%-3%; although somewhat higher in the betamethasone animals, none of the between or within group differences were significant. CONCLUSION: Fetal cerebral metabolism in the preterm ovine fetus is altered by antenatal glucocorticoid administration, which is comparable with that used in human pregnancy, and includes an acute decrease in cerebral blood flow and a probable increase in anaerobic glucose metabolism. Although likely of short duration in conjunction with peak glucocorticoid levels, these metabolic effects may place the developing brain at added risk for superimposed hypoxic injury.

Effects of antenatal glucocorticoids on cerebral protein synthesis in the preterm ovine fetus.

OBJECTIVE: Although antenatal glucocorticoids have well-known benefits for infants who are born preterm by the enhancement of pulmonary maturation, adverse effects on brain growth and development have been reported in several animal-based studies. We have used the chronically catheterized ovine fetus to determine the effects of synthetic glucocorticoids that are administered at doses used clinically on cerebral protein synthesis during early brain development using [13C]-leucine tracer method. STUDY DESIGN: Chronically instrumented pregnant sheep at 0.85 gestation received 2 intramuscular injections of betamethasone at 170 microg/kg maternal weight or saline 24 hours apart together with a continuous infusion of L-[1-(13)C]-leucine to the fetus on the second day of experimentation. Measurements were obtained for fetal plasma leucine enrichment at steady-state and brain tissue intracellular free and protein-bound leucine enrichment at necropsy, followed by the determination of cerebral protein fractional synthetic rates (FSRs). A coefficient of variation was determined for plasma and tissue enrichment measurements to assess the inherent methodologic variance with the use of [13C]-leucine tracer technology. RESULTS: The cerebral protein FSR averaged approximately 112% per day and approximately 35% per day when the intracellular free and plasma enrichment values were used for the precursor pool measurements, respectively, providing for maximal and minimal FSR values. There were no differences between cortical and cerebellar tissues nor between the saline control and the betamethasone animal groups. The coefficient of variation for the plasma-enrichment values averaged approximately 2%; the coefficient of variation for the tissue enrichment values averaged approximately 10%, with an inverse relationship between the [13C]-leucine enrichment values and the coefficient of variation values. CONCLUSION: Although cerebral FSR values for the preterm ovine fetus are high and indicate high rates of protein synthesis and degradation, we found no evidence that these are altered by betamethasone as used clinically and thereby do not account for the reported structural alterations in the brain after a single-course of antenatal glucocorticoids.

Correction: Cerebral microbleeds in a neonatal rat model.

[This corrects the article DOI: 10.1371/journal.pone.0171163.].

Effect of hypobaria and hyperoxia during sepsis on survival and energy metabolism.

BACKGROUND: Injured warfighters air evacuated to tertiary medical care facilities are subjected to many stresses that may promote the development of sepsis. In this study, we tested the hypothesis that exposure to "in-flight" hypobaria and/or hyperoxia within 24 hours after onset of intra-abdominal infection in rats accelerates the development and/or severity of sepsis and neurologic injury in survivors. METHODS: Sprague-Dawley rats underwent cecal ligation/puncture (CLP) or sham procedures. Twenty-four hours later, rats were then placed in hypobaric chambers for 6 hours and assigned to normobaric conditions and maintained at either 21% or 100% O2, or under hypobaric conditions (pressure equivalent to an altitude of 8,000 ft) but maintained under either 28% or 100% O2. Two days after CLP or sham, blood samples were obtained for cytokine levels, and mitochondria were isolated from the brain and heart of a subset of animals for analysis of mitochondrial oxygen consumption. Animals were also evaluated for neuromotor impairment before and 15 days postsurgery. RESULTS: Among the 70 rats studied, 16.7% of CLP but none of the sham-treated rats died. All of the CLP but none of the sham rats had evidence of peritonitis at 2 days. Twenty percent (6 of 30) CLP rats undergoing hypobaria versus 12.5% (3 of 24) of CLP rats exposed to normobaria died (p = 0.715) while 12% (3 of 25) of CLP rats exposed to hyperoxia versus 20.7% (6 of 29) of CLP rats exposed to normoxia died (p = 0.48). The ratio of mitochondrial ATP-generating O2 consumption to resting respiration was higher in the CLP plus hypobaria under 100% compared with shams. The only difference in H2O2 production was observed in mitochondria from CLP rats exposed to hyperoxia under normobaric conditions. Composite neurologic scores obtained 15 days postinjury were lower than those at baseline for shams. CONCLUSION: We conclude that neither "in-flight" hyperoxia nor hypobaria exacerbate sepsis or neurologic injury.

Cerebral microbleeds in a neonatal rat model.

BACKGROUND: In adult humans, cerebral microbleeds play important roles in neurodegenerative diseases but in neonates, the consequences of cerebral microbleeds are unknown. In rats, a single pro-angiogenic stimulus in utero predisposes to cerebral microbleeds after birth at term, a time when late oligodendrocyte progenitors (pre-oligodendrocytes) dominate in the rat brain. We hypothesized that two independent pro-angiogenic stimuli in utero would be associated with a high likelihood of perinatal microbleeds that would be severely damaging to white matter. METHODS: Pregnant Wistar rats were subjected to intrauterine ischemia (IUI) and low-dose maternal lipopolysaccharide (mLPS) at embryonic day (E) 19. Pups were born vaginally or abdominally at E21-22. Brains were evaluated for angiogenic markers, microhemorrhages, myelination and axonal development. Neurological function was assessed out to 6 weeks. RESULTS: mRNA (Vegf, Cd31, Mmp2, Mmp9, Timp1, Timp2) and protein (CD31, MMP2, MMP9) for angiogenic markers, in situ proteolytic activity, and collagen IV immunoreactivity were altered, consistent with an angiogenic response. Vaginally delivered pups exposed to prenatal IUI+mLPS had spontaneous cerebral microbleeds, abnormal neurological function, and dysmorphic, hypomyelinated white matter and axonopathy. Pups exposed to the same pro-angiogenic stimuli in utero but delivered abdominally had minimal cerebral microbleeds, preserved myelination and axonal development, and neurological function similar to naïve controls. CONCLUSIONS: In rats, pro-angiogenic stimuli in utero can predispose to vascular fragility and lead to cerebral microbleeds. The study of microbleeds in the neonatal rat brain at full gestation may give insights into the consequences of microbleeds in human preterm infants during critical periods of white matter development.

Kinetics of betamethasone and fetal cardiovascular adverse effects in pregnant sheep after different doses.

OBJECTIVE: To study the pharmacokinetics of different betamethasone doses and preparations used to enhance fetal lung maturation in the maternal and fetal circulation of sheep and the adverse effects on fetal blood pressure. METHODS: Doses of 170 (n = 6) and 110 microg/kg (n = 6) betamethasone phosphate equivalent to 12 or 8 mg, respectively, administered to a 70 kg pregnant woman or 170 microg/kg (n = 6) of a depot formulation (50% betamethasone phosphate and 50% betamethasone acetate) were injected intramuscularly to chronically instrumented pregnant sheep. RESULTS: Both betamethasone preparations produced highest maternal concentrations after 15 min followed by an exponential decline with a t(1/2) of about 3 hours. The drug fell below the limit of detection at 8 to 12 hours. Betamethasone was first detectable in the fetal circulation at 1 hour, peaked at 3 hours, and decreased below the limit of detection at 8 hours independently of the dose or preparation. Maternal and fetal betamethasone concentrations achieved with the phosphate and acetate formulation were one half of those obtained with betamethasone phosphate, suggesting that very little betamethasone is released from the acetate within the first 8 hours when the effect on lung maturation is needed. Betamethasone led to a maximal increase of mean fetal blood pressure from 42+/-1 to 51+/-1 mm Hg (P < .05) and did not differ between the doses and preparations, although plasma concentrations showed a clear dose-concentration relationship. CONCLUSION: The doses of betamethasone used in obstetrics are supramaximal in terms of cardiovascular effects in sheep. Risk-benefit studies are needed to find the effective steroid dose with the least adverse effects.

Betamethasone effects on ovine uterine and umbilical placental perfusion at the dose used to enhance fetal lung maturation.

OBJECTIVE: The purpose of this study was to examine glucocorticoid effects on umbilical placental perfusion. STUDY DESIGN: Pregnant sheep instrumented with uterine and umbilical ultrasound transit-time flow probes received 2 doses of 12 mg betamethasone (n = 6) or saline (n = 5) intramuscularly 24 hours apart. RESULTS: Maternal blood pressure and uterine flow did not change during glucocorticoid exposure. Fetal blood pressure increased, and umbilical resistance showed a transient increase after each injection (P < .05), followed by an increase of umbilical flow (P < .05) that was closely correlated to an increase in fetal heart rate (r = 0.85, P < .001), which determines cardiac output of the developing heart. Umbilical waveform indices were decreased over the entire treatment period, indicating a decrease of resistance in the fetoplacental microcirculation (P < .05). CONCLUSION: Fetoplacental perfusion during glucocorticoid exposure is not limiting for nutrition exchange. Clinical interpretation of Doppler waveform indices needs to be interpreted with caution because they do not reflect dynamics of umbilical placental perfusion.

Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age.

The experiments reported here were designed for both in vivo and in vitro approaches in the same animals to obtain a better picture of the role of estrogen in the control of parturition. Chronically catheterized pregnant ewes were treated with vehicle (n = 5) or estradiol (n = 6), 5 mg twice a day, im for 2 d starting at d 119 of gestation. Maternal and fetal plasma estradiol, progesterone, and cortisol were measured by RIA and maternal plasma prostaglandin (PG) F2alpha was measured by enzyme immunoassay. Intrauterine PG H synthase 2 mRNA and protein and placental P450(c17)alpha hydroxylase mRNA were determined by Northern, in situ hybridization, Western blot analysis, and immunocytochemistry. Data were analyzed by ANOVA. Five of six estradiol-treated ewes delivered their fetuses within 48 h; however, the placenta was still retained 5-6 h after fetal delivery. Both maternal plasma estradiol and PGF2 alpha increased significantly in the estradiol-treated group. Maternal and fetal plasma progesterone and cortisol were not altered in either group. There were significant increases of PGH synthase 2 mRNA and protein in myometrium, endometrium, and maternal placenta but not in fetal placenta in estradiol-treated ewes. Placental P450(c17)alpha hydroxylase mRNA was not detectable in vehicle or estradiol-treated groups. Estradiol can, in the absence of increase in plasma cortisol, stimulate uterine PG production and induce labor, resulting in fetal delivery in the sheep. Failure of placental delivery after estradiol treatment suggests that estradiol alone is insufficient to stimulate some of the key changes required to complete delivery at the stage of gestation studied.

Effects of maternally administered dexamethasone and acute hypoxemia at 0.7 gestation on blood pressure and placental perfusion in sheep.

Glucocorticoid administration to women in premature labor significantly decreases preterm infant morbidity and mortality. Fetal exposure to maternally administered glucocorticoids in late gestation causes fetal hypertension. We determined the effects of a single course (4 injections at 12-hr intervals) of dexamethasone (DM; 2 mg, a weight-adjusted dose equivalent to one-third the dose administered to pregnant women) or saline (S) in sheep at 103-104 days of gestation (dGA; term 149 dGA) on maternal and fetal blood pressure (BP). We also determined the BP and placental perfusion effects of acute maternal hypoxemia. Venous and arterial catheters were placed in 10 ewes and fetuses (DM = 6, S = 4) at 96 +/- 1 dGA. Maternal and fetal placental perfusion was determined with fluorescent microspheres. Dexamethasone increased fetal but not maternal BP; maternal and fetal placental blood flow and vascular resistance (VR) were unchanged. At 105 dGA, hypoxemia was induced for 1 hr by maternal nitrogen gas inhalation to decrease fetal PaO2 by 40%. Hypoxemia increased BP in DM but not S fetuses or mothers in either group. Hypoxemia decreased maternal placental blood flow by 39 +/- 7% and 51 +/- 9% and increased maternal placental VR by 65 +/- 7% and 69 +/- 6% in S and DM mothers, respectively. Hypoxemia did not alter fetal placental blood flow or VR in either treatment group. In summary, at 0.7 gestation, DM induces a hypertensive response to fetal hypoxemia that is characteristic of older fetuses but does not alter hypoxemia-induced reductions in maternal placental blood flow.

Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy.

Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-ß-cyclodextrin, methyl-ß-cyclodextrin, and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-ß-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.

The protective effect of glibenclamide in a model of hemorrhagic encephalopathy of prematurity.

We studied a model of hemorrhagic encephalopathy of prematurity (EP) that closely recapitulates findings in humans with hemorrhagic EP. This model involves tandem insults of 20 min intrauterine ischemia (IUI) plus an episode of elevated venous pressure induced by intraperitoneal glycerol on post-natal day (P) 0. We examined Sur1 expression, which is upregulated after focal ischemia but has not been studied after brief global ischemia including IUI. We found that 20 min IUI resulted in robust upregulation of Sur1 in periventricular microvessels and tissues. We studied tandem insult pups from untreated or vehicle-treated dams (TI-CTR), and tandem insult pups from dams administered a low-dose, non-hypoglycemogenic infusion of the Sur1 blocker, glibenclamide, for 1 week after IUI (TI-GLIB). Compared to pups from the TI-CTR group, pups from the TI-GLIB group had significantly fewer and less severe hemorrhages on P1, performed significantly better on the beam walk and accelerating Rotarod on P35 and in tests of thigmotaxis and rapid learning on P35-49, and had significantly greater body and brain weights at P52. We conclude that low-dose glibenclamide administered to the mother at the end of pregnancy protects pups subjected to IUI from post-natal events of elevated venous pressure and its consequences.

Tandem insults of prenatal ischemia plus postnatal raised intrathoracic pressure in a novel rat model of encephalopathy of prematurity.

OBJECT: Encephalopathy of prematurity (EP) is common in preterm, low birth weight infants who require postnatal mechanical ventilation. The worst types of EP are the hemorrhagic forms, including choroid plexus, germinal matrix, periventricular, and intraventricular hemorrhages. Survivors exhibit life-long cognitive, behavioral, and motor abnormalities. Available preclinical models do not fully recapitulate the salient features of hemorrhagic EP encountered in humans. In this study, the authors evaluated a novel model using rats that featured tandem insults of transient prenatal intrauterine ischemia (IUI) plus transient postnatal raised intrathoracic pressure (RIP). METHODS: Timed-pregnant Wistar rats were anesthetized and underwent laparotomy on embryonic Day 19. Intrauterine ischemia was induced by clamping the uterine and ovarian vasculature for 20 minutes. Natural birth occurred on embryonic Day 22. Six hours after birth, the pups were subjected to an episode of RIP, induced by injecting glycerol (50%, 13 µl/g intraperitoneally). Control groups included naive, sham surgery, and IUI alone. Pathological, histological, and behavioral analyses were performed on pups up to postnatal Day 52. RESULTS: Compared with controls, pups subjected to IUI+RIP exhibited significant increases in postnatal mortality and hemorrhages in the choroid plexus, germinal matrix, and periventricular tissues as well as intraventricularly. On postnatal Days 35-52, they exhibited significant abnormalities involving complex vestibulomotor function and rapid spatial learning. On postnatal Day 52, the brain and body mass were significantly reduced. CONCLUSIONS: Tandem insults of IUI plus postnatal RIP recapitulate many features of the hemorrhagic forms of EP found in humans, suggesting that these insults in combination may play important roles in pathogenesis.

Escherichia coli O157:H7 infection in Dutch belted and New Zealand white rabbits.

Enterohemorrhagic Escherichia coli (EHEC) produce one or more types of Shiga toxins and are foodborne causes of bloody diarrhea. The prototype EHEC strain, Escherichia coli O157:H7, is responsible for both sporadic cases and serious outbreaks worldwide. Infection with E. coli that produce Shiga toxins may lead to diarrhea, hemorrhagic colitis, or (less frequently) hemolytic uremic syndrome, which can cause acute kidney failure. The exact mechanism by which EHEC evokes intestinal and renal disease has not yet been determined. The development of a readily reproducible animal oral-infection model with which to evaluate the full pathogenic potential of E. coli O157:H7 and assess the efficacy of therapeutics and vaccines remains a research priority. Dutch belted (DB) rabbits are reported to be susceptible to both natural and experimental EHEC-induced disease, and New Zealand white (NZW) rabbits are a model for the intestinal manifestations of EHEC infection. In the current study, we compared the pathology caused by E. coli O157:H7 infection in DB and NZW rabbits. Both breeds of rabbits developed clinical signs of disease and intestinal lesions after experimental infection. In addition, one of the infected DB rabbits developed renal lesions. Our findings provide evidence that both breeds are susceptible to E. coli O157:H7 infection and that both may be useful models for investigating EHEC infections of humans.

Diagnosis and prevention of dissemination of tuberculosis in a recently imported rhesus macaque (Macaca mulatta).

Thirty-four (34) days after arrival at our facility, a recently imported rhesus macaque demonstrated a grade 4/5 reaction to intradermal testing with mammalian old tuberculin and a strong positive response in a serum interferon gamma (IFN-gamma) stimulation assay (Primagam). The affected animal was euthanized to prevent further exposure of the other rhesus in the quarantine room. Necropsy revealed enlarged, caseating mediastinal lymph nodes. Further analysis confirmed the presence of Mycobacterium tuberculosis complex organisms. Strict isolation measures were initiated and intensive testing of all animals in the affected room began immediately. After 13 weeks of additional testing, none of the animals in the room showed any positive response and all were released from quarantine. This case illustrates the importance of prolonged quarantine of non-human primates (NHP) and illustrates the usefulness of serology-based diagnostics as an adjunct to intradermal testing (molecular-based diagnostics typically refers to polymerase chain reaction, whereas this assay is really serology based, even though it is an in vitro IFN-gamma stimulation assay). It also demonstrates that with proper isolation procedures, the spread of tuberculosis can be prevented in NHP facilities.