Newborn Guinea Pigs Research Articles

Dose–Response Effects of Glutathione Supplement in Parenteral Nutrition on Pulmonary Oxidative Stress and Alveolarization in Newborn Guinea Pig

In premature infants, glutathione deficiency impairs the capacity to detoxify the peroxides resulting from O2 metabolism and those contaminating the parenteral nutrition (PN) leading to increased oxidative stress, which is a major contributor to bronchopulmonary dysplasia (BPD) development. In animals, the supplementation of PN with glutathione prevented the induction of pulmonary oxidative stress and hypoalveolarization (characteristic of BPD). Hypothesis: the dose of glutathione that corrects the plasma glutathione deficiency is sufficient to prevent oxidative stress and preserve pulmonary integrity. Three-day-old guinea pigs received a PN, supplemented or not with GSSG (up to 1300 µg/kg/d), the stable form of glutathione in PN. Animals with no handling other than being orally fed constituted the control group. After 4 days, lungs were removed to determine the GSH, GSSG, redox potential and the alveolarization index. Total plasma glutathione was quantified. The effective dose to improve pulmonary GSH and prevent the loss of alveoli was 330 µg/kg/d. A 750 µg/kg/d dose corrected the low-plasma glutathione, high-pulmonary GSSG and oxidized redox potential. Therefore, the results suggest that, in a clinical setting, the dose that improves low-plasma glutathione could be effective in preventing BPD development.

Pharmacological Prevention of Neonatal Opioid Withdrawal in a Pregnant Guinea Pig Model.

Newborns exposed to prenatal opioids often experience intense postnatal withdrawal after cessation of the opioid, called neonatal opioid withdrawal syndrome (NOWS), with limited pre- and postnatal therapeutic options available. In a prior study in pregnant mice we demonstrated that the peripherally selective opioid antagonist, 6β-naltrexol (6BN), is a promising drug candidate for preventive prenatal treatment of NOWS, and a therapeutic mechanism was proposed based on preferential delivery of 6BN to fetal brain with relative exclusion from maternal brain. Here, we have developed methadone (MTD) treated pregnant guinea pigs as a physiologically more suitable model, enabling detection of robust spontaneous neonatal withdrawal. Prenatal MTD significantly aggravates two classic maternal separation stress behaviors in newborn guinea pigs: calling (vocalizing) and searching (locomotion) - natural attachment behaviors thought to be controlled by the endogenous opioid system. In addition, prenatal MTD significantly increases the levels of plasma cortisol in newborns, showing that cessation of MTD at birth engages the hypothalamic-pituitary-adrenal (HPA) axis. We find that co-administration of 6BN with MTD prevents these withdrawal symptoms in newborn pups with extreme potency (ID50 ∼0.02 mg/kg), at doses unlikely to induce maternal or fetal withdrawal or to interfere with opioid antinociception based on many prior studies in rodents and non-human primates. Furthermore, we demonstrate a similarly high potency of 6BN in preventing opioid withdrawal in adult guinea pigs (ID50 = 0.01 mg/kg). This high potency appears to run counter to our pharmacokinetic studies showing slow 6BN transit of both the placenta and maternal blood brain barrier in guinea pigs, and calls into question the preferential delivery mechanism. Rather, it suggests a novel receptor mechanism to account for the selectively high potency of 6BN to suppress opioid dependence at all developmental stages, even in adults, as compared to its well-established low potency as a classical opioid antagonist. In conclusion, 6BN is an attractive compound for development of a preventive therapy for NOWS.

Relationship between redox potential of glutathione and DNA methylation level in liver of newborn guinea pigs

ABSTRACT The metabolism of DNA methylation is reported to be sensitive to oxidant molecules or oxidative stress. Hypothesis: early-life oxidative stress characterized by the redox potential of glutathione influences the DNA methylation level. The in vivo study aimed at the impact of modulating redox potential of glutathione on DNA methylation. Newborn guinea pigs received different nutritive modalities for 4 days: oral nutrition, parenteral nutrition including lipid emulsion Intralipid (PN-IL) or SMOFLipid (PN-SF), protected or not from ambient light. Livers were collected for biochemical determinations. Redox potential (p < 0.001) and DNA methylation (p < 0.01) were higher in PN-infused animals and even higher in PN-SF. Their positive correlation was significant (r2 = 0.51; p < 0.001). Methylation activity was higher in PN groups (p < 0.01). Protein levels of DNA methyltransferase (DNMT)-1 were lower in PN groups (p < 0.01) while those of both DNMT3a isoforms were increased (p < 0.01) and significantly correlated with redox potential (r2 > 0.42; p < 0.001). The ratio of SAM (substrate) to SAH (inhibitor) was positively correlated with the redox potential (r2 = 0.36; p < 0.001). In conclusion, early in life, the redox potential value strongly influences the DNA methylation metabolism, resulting in an increase of DNA methylation as a function of increased oxidative stress. These results support the notion that early-life oxidative stress can reprogram the metabolism epigenetically. This study emphasizes once again the importance of improving the quality of parenteral nutrition solutions administered early in life, especially to newborn infants. Abbreviation of Title: Parenteral nutrition and DNA methylation

OXIDATIVE STRESS MARKERS IN THE LUNGS UNDER EXPERIMENTAL HYPEROXIA WITH INHALED LIPOSOMES BASED ON EGG LECITHIN

Oxidative stress is considered to be a factor leading to lung damage in premature infants. The aim of this study was to investigate the effect of inhaled antioxidants incorporated into egg lecithin liposomes on the indicators of oxidative stress in the lungs of newborn guinea pigs under experimental hyperoxia (3 days). Bronchoalveolar lavage fl uid (BALF) was used as a material for the study. Under hyperoxia exposure, inhalation of liposomes containing N-acetylcysteine and alpha-tocopherol contributed to the suppression of the reactive oxygen species production by cells, normalization of glutathione peroxidase activity and carbonyls content, while not affecting the level of diene conjugates in BALF. The introduction of retinoid-containing liposomes (retinol and retinoic acid) under hyperoxia was accompanied by normalization of glutathione peroxidase activity as well as the content of protein oxidation products in BALF, while the generation of reactive oxygen species remained enhanced, and the diene conjugates and thiobarbituric acid reactive products exceeded the levels in animals exposed to hyperoxia alone. Thus, the inhaled liposomes containing retinoids and egg lecithin exhibit not only anti-, but also a prooxidant effect in the lungs under hyperoxia exposure, unlike the liposomal forms of N-acetylcysteine and alpha-tocopherol.

An arterially perfused brainstem preparation of guinea pig to study central mechanisms of airway defense

The perfused working heart brainstem preparation of rodents has become a widely used tool to study brainstem function. Here, we adapt this experimental technique for newborn guinea pigs (postnatal day 7–14) to develop a tool that enables investigation of airway defense mechanisms not observed in other rodents. The perfused guinea pig brainstem preparation generates a stable eupnea-like motor pattern recorded from the phrenic, recurrent laryngeal and intercostal nerves and basic cardio-respiratory reflexes, including the arterial chemoreceptor, the baroreceptor reflex. In addition a fictive laryngeal cough reflex can be reliably elicited after mechanical stimulation of the trachea. Single unit recordings within the ponto-medullary respiratory column show robust central respiratory neuronal activity. Additionally, as in other species ponto-medullary transection of the brainstem produces apneusis. The latter suggests that the preparation fully preserves ponto-medullary synaptic connectivity that is required for eupnea-like respiratory rhythm and pattern formation and the mediation of various cardio-respiratory reflexes. We conclude that this novel research tool provides an alternative to established rat and mouse preparations and may become a experimental tool for the investigation of central mechanisms that mediate laryngeal cough.

GLUTATHIONE ADDITION TO PARENTERAL NUTRITION PREVENTS PULMONARY OXIDATIVE STRESS AND HYPO-ALVEOLARIZATION IN NEWBORN GUINEA PIG

Abstract BACKGROUND Parenteral nutrition (PN) is contaminated by peroxides, which induce oxidative stress (oxidized redox potential) and hypoalveolarization in lungs of newborn animals. In human premature newborns, PN is associated to an oxidized redox potential and to a greater prevalence of bronchopulmonary dysplasia (BPD). Peroxides are detoxified by glutathione that is deficient in preterm infants. Hypothesis: Improving glutathione level in premature infants prevents BPD development caused by oxidative stress. OBJECTIVES Assess dose-response effect of glutathione added in PN on alveolarization index and pulmonary redox potential of glutathione (specific oxidative stress marker for peroxides) in newborn guinea pigs. DESIGN/METHODS At day 3 of life, through a jugular catheter, guinea pigs (N=55) received PN (dextrose, amino acids, lipids, vitamins, electrolytes) enriched in glutathione (0, 50, 120, 165, 270, 370, 650 μg GSSG/kg/d). GSSG was used rather than GSH because it has a better stability in PN and similar efficiency in vivo. A control group (no manipulation, fed by mouth) served as reference. After 4 days, lungs were taken for determination of GSH, GSSG, redox potential (capillary electrophoresis) and alveolarization index (AI) (number of intercepts between a 1 mm line and histological structures); higher is AI, higher is the number of alveoli. Data (mean±sem) from animals on PN were compared by ANOVA, p&lt;0.05. RESULTS There was no difference in the alveolarization index between groups receiving doses ranging from 0 to 120 μg/kg/d (25.9 ± 0.6 intercepts/mm) and between those with doses ranging between 165 and 650 μg/kg/d (30.5 ± 0.6 intercepts/mm). However, the difference between the lowest (0–120 μg/kg/d) and the highest doses (165–650 μg/kg/d) was highly significant (p&lt;0.001). The control AI value was 29.4 ± 1.2 intercepts/mm. Increasing GSSG in PN has allowed a significant (r2=0.47; p&lt;0.001) reduction of redox potential, from oxidized status of -205 ± 2 in PN without GSSG to -215 ± 2 mV with the highest dose (650 μg/kg/d); control = -222 ± 3 mV. GSSG in PN was also associated with a decreasing GSSG values (r2=0.49; p&lt;0.001), from 0.84 ± 0.10 in PN without GSSG to 0.38 ± 0.06 nmol/mg prot with the highest dose (650 μg/kg/d); control = 0.35 ± 0.07 nmol/mg prot. There was no effect on GSH (mean value of 29 ± 1 nmol/mg prot). CONCLUSION Addition of glutathione into PN prevents pulmonary oxidative stress. The dose-response curve suggests a specific minimal dose of GSSG to maintain the alveolar integrity. Lower doses were inefficient whereas higher doses are unnecessary, as the index of alveolarization reached a plateau with a mean value similar to that one observed in the control group. This pre-clinical study brings basic data that will serve for the clinical phase I-II study that we are starting soon.

Impact of SMOFLipid on Pulmonary Alveolar Development in Newborn Guinea Pigs.

Parenteral nutrition (PN) is associated with bronchopulmonary dysplasia in premature infants. In animals, PN leads to alveolar loss following stimulation of apoptosis by oxidative stress (oxidized redox potential). Peroxides and aldehydes generated in PN can induce hypo-alveolarization. The implication of peroxides, which is reduced by light protection, is demonstrated. The implication of aldehydes from omega-6 fatty acids oxidation is expected. The hypothesis is that composition and light exposure of PN influences bronchopulmonary dysplasia development. Since SMOFLipid (SMOF) contains a lower amount of omega-6 fatty acids than Intralipid (IL), the aim was to compare, the impacts of PN compounded with SMOF or IL, photo-protected or not, on alveolar development. Three-day-old Guinea pigs received PN, photo-protected or not, made with SMOF or IL through a jugular vein catheter. After 4 days, lungs were sampled for determinations of redox potential of glutathione, apoptosis (caspase-3, caspase-8, and caspase-9) and alveolarization index (histology: number of intercepts/mm). Compared with IL, SMOF induces a greater oxidation of redox potential (-200 ± 1 versus [vs] -205 ± 1 mV), apoptosis (caspase-3: 0.27 ± 0.04 vs 0.16 ± 0.02; caspase-9: 0.47 ± 0.03 vs 0.30 ± 0.03), and a lower alveolarization index (27.2 ± 0.8 vs 30.0 ± 0.9). Photo-protection prevented activation of caspase-9 and was statistically without effect on redox potential, caspase-3, and alveolarization index. In our model, SMOF is pro-oxidant and induces hypo-alveolarization following exaggerated apoptosis. These results highlight the need for further studies before introducing SMOFLipid in standard neonatal care.

191 - Dose-response Effect of Glutathione added in Parenteral Nutrition on Lung Oxidative Stress and Alveolarization in Newborn Guinea Pig

Peroxides contaminating parenteral nutrition (PN) are associated with oxidized redox potential of glutathione in blood of preterm newborns ( Methods At day 3 of life, through a jugular catheter, guinea pigs (N=55) received PN (dextrose, amino acids, lipids, vitamins, electrolytes) enriched in glutathione (0, 50, 120, 165, 270, 370, 650 μg GSSG/kg/d). A control group (no manipulation, fed by mouth) served as reference. After 4 days, lungs were taken for determination of GSH, GSSG, redox potential (capillary electrophoresis) and alveolarization index (AI) (number of intercepts between a 1 mm line and histological structures). Data (mean ± sem) from animals on PN were compared by ANOVA, p Results The low AI observed with low doses (μg/kg/d) of GSSG (0-120: 26±1) was corrected with higher level (165-650: 30±1, p Conclusion Addition of glutathione into PN allows detoxification of peroxides (lower GSSG), preventing oxidation of redox and loss of alveoli. Our team is funded to start a clinical study soon.

Long-term Impact of added Glutathione into Parenteral Nutrition Administered During the First week of life on Redox Potential and Energy Metabolism Enzymes in Guinea pig liver

Introduction Peroxides are generated in parenteral nutrition (PN). Infusion of PN or H2O2 in the first week of life of animals alters later in life their hepatic energy metabolism. Glutathione addition in PN prevented pulmonary oxidative stress induced by PN in newborn guinea pigs (GP). Hypothesis: Addition of glutathione into PN prevents the long-term impact of PN on activities of key enzymes of energy metabolism (glucokinase (GK), phosphofructokinase (PFK) and acetyl-CoA carboxylase (ACC)). Methods 4 groups (N=48) of 3-day old GP were used. Control: no manipulation, fed ad libitum all time; PN: animals nourished exclusively with a complete PN via a catheter; PN+ 6 or 12 µM GSSG; GSSG was used as pro-GSH. After 4 days, hepatic redox potential was measured in half of GP. The other half started oral diet. At 16 weeks of age, liver was collected for determination of GSH, GSSG and redox potential (capillary electrophoresis, Nernst’s equation), activities (U: nmol/min/mg prot) of PFK, GK (calorimetric method), and ACC (method with radioactive tracer). Data (mean±sem) were analyzed by ANOVA, p Results Immediately after treatments, there was no difference between groups for redox potential (mV) (-231±1). Four months later, redox potential, GSH and GSSG (nmol/mg prot) were lower in PN groups independently of GSSG addition, relatively to control (respectively, PN groups: -235±1, 69±2, 0.8±0.1 vs. Control: -229±2, 81±5, 1.8±0.3). ACC activity was higher in PN group (4.5±0.3 U) and greater in NP+GSSG groups (5.7±0.3 U) relatively to control (3.2±0.2 U). GK and PFK activities did not differ. Conclusion PN early in life induces later in liver reduction of redox potential and higher activity of a key enzyme of lipogenesis (ACC). In contrast to beneficial effect previously observed in lungs, addition of GSSG into PN did not prevent the long-term effects of PN on the liver.

Studies on vertical transmission of Trichinella spiralis in experimentally infected guinea pigs (Cavia porcellus).

An experimental study to enhance knowledge on the capability of Trichenella spiralis to pass from guinea pigs to progeny at different periods of pregnancy or lactation was performed. For this purpose, 18 female adult guinea pigs were inoculated with 100 or 1000 T. spiralis muscle larvae (ML) during early, late gestation and during lactation period. The presence of T. spiralis (ML) in mothers and newborns was studied through enzymatic digestion from muscle samples. ML were observed in 9 of 42 newborn guinea pigs and levels of infection were significantly higher when infections of mothers were done during late gestation (p=0.0046) with the high infective dose (p=0.0043). T. spiralis ML were not recovered from any of the newborns from mothers infected in the lactation period. Ten out of 18 infected mothers presented larvae 1 in their mammary glands. Muscle samples from the tongue and the masseter showed the highest larval burdens. These observations confirm previous reports on that ML of T. spiralis are capable to pass through placental tissues to reach and encyst in striated muscle groups of newborn guinea pigs. This study may also reinforce the importance of preventive programs to control trichinellosis in those endemic areas where pregnant women would have high risk of infection.

Correction of the oxidant-antioxidant balance in lungs during hyperoxia by liposomal alpha-tocopherol and retinoids in the experiment

The influence of inhaled liposomes, containing dipalmitoyl phosphatidylcholine and a-tocopherol, and liposomes containing dipalmitoyl phosphatidylcholine, retinol and retinoic acid, on parameters of the oxidantantioxidant system in lungs of newborn guinea pigs exposed to hyperoxia during 3 and 14 days has been studied. Administration of both types of liposomes under conditions of prolonged hyperoxia (14 days) results in normalization of glutathione peroxidase activity and prevents elevation of the levels of lipid and protein peroxidation products in bronchoalveolar lavage fluid. Unlike liposomes with a-tocopherol, administration of liposomes containing retinoids did not cause the normalizing effect on the content of nonprotein SH-compounds in the bronchoalveolar fluid and contributed to significant reduction of the a-tocopherol level in lung tissues.

154 - Effect of Parenteral Lipid Emulsion SMOF Lipid and Intralipid on Redox Potential of Glutathione and DNA Methylation in Newborn Guinea Pig

Infants born Objectives To compare the oxidative property of SM and IL by measuring redox potential of glutathione, and to correlate the DNA methylation values with the redox values. Methods At three day of life, guinea pigs received PN (dextrose, amino acids, vitamins, electrolytes) compounded with SM (n=15) or IL (n=16) through a jugular vein catheter. After 4 days of infusion, redox potential of glutathione (mV, by the Nernst equation using the GSH and GSSG concentrations measured by capillary electrophoresis) and global DNA methylation (U = nmol of 5MedCyt / μg DNA; commercial kit) were determined in liver. Statistics: t test and Pearson correlation; p Results The mean (±sem) redox potential was higher in SM (SM: -207±1, IL: -213±2 mV, p -1 •x – 2.96U; r 2 = 0.37, p Conclusion In our animal model, compared to IL, SM is a more potent oxidizing agent, which could have important impact on health throughout life.

Culture, characterization and differentiation of neural precursors from the central nervous system of guinea pigs (Cavia porcellus Linnaeus, 1758)

Abstract: Potentially neurogenic areas were initially identified by incorporation of bromodeoxyuridine (BrdU) in cells underlying the subventricular zone (SVZ) of the lateral ventricles wall, hippocampus and olfactory bulbs of newborn guinea pigs. Neural precursors from the SVZ were cultured in suspension, generating neurospheres (NSFs), which, upon dissociation were able to generate new NSFs. Upon culture in the absence of growth factors, cells dissociated from NSFs displayed evidence for neural differentiation, giving rise to cells from neural lineage. Flow cytometry analysis for of NSFs-derived cells after differentiation revealed approximately 13.3% nestin positive, 5.5% Beta-III-tubulin positive, 9% GFAP positive and 7.8% mGalC positive. Functional assays by measurement of calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli, revealed stimulation in differentiated cells, an indicator of neuronal differentiation. The ability of guinea pig SVZ cells to originate functional neurons in vitro is promising for research and towards a future use of neural stem cells in the therapy of neurological disorders.

Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity

BackgroundThe oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. AimTo investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. MethodsThree days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180μM ascorbylperoxide; (6) D+350μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1mM DTT. Data were compared by ANOVA, p<0.05. ResultsMAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. ConclusionThe present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity.

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model.

Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated Δ145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 10(7) PFU of Δ145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 10(5) or 10(6) PFU of Δ145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. Δ145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with Δ145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 10(6) PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. Previous attempts to develop successful immunization against cytomegalovirus have largely centered on subunit vaccination against virion proteins but have yielded disappointing results. The advent of bacterial artificial chromosome technologies has enabled engineering of recombinant cytomegaloviruses (CMVs) from which virus genome-encoded immune modulation genes have been deleted, toward the goal of developing a safe and potentially more efficacious live attenuated vaccine. Here we report the findings of studies of such a vaccine against congenital CMV infection based on a virus with a targeted deletion in gp145, a virus genome-encoded inhibitor of protein kinase R, using the guinea pig model of vertical CMV transmission. The deletion virus was attenuated for dissemination in immunocompromised guinea pigs but elicited ELISA and neutralizing responses. The vaccine conferred protection against maternal DNAemia and congenital transmission and resulted in reduced viral loads in newborn guinea pigs. These results provide support for future studies of attenuated CMV vaccines.

Expression of P2X3 and P2X 5 myenteric receptors varies during the intestinal postnatal development in the guinea pig.

P2X3 receptor expression in various tissues appears to be modulated by age. In the present study, we used single cell RT-PCR to determine the number of P2X3 positive myenteric neurons at different stages of guinea pig postnatal development, and we tested if similar changes also occur to other myenteric P2X receptors. Moreover, we carried out whole-cell recordings using Patch Clamp techniques to determine possible changes in P2X receptors sensitivity to ATP and α,β-methylene ATP (α,β-meATP) between newborn and adult animals. Our data indicate that P2X3 subunit transcripts are present in a larger number of myenteric neurons from newborn guinea pigs whereas P2X5 mRNA is found more frequently in adults. Expression of P2X2 and P2X4 transcripts does not change during postnatal development. In newborn animals, virtually all neurons expressing P2X3 also expressed P2X2 transcripts. This is important because these two subunits are known to form heteromeric channels. ATP potency to activate P2X receptors in neurons of both newborn and adult animals was the same. α,β-meATP, a known P2X3 receptor agonist, induces only a marginal current despite the fact of the higher presence of P2X3 subunits in newborns. These findings imply that P2X3 subunits are mainly forming heteromeric, α,β-meATP insensitive channels perhaps because P2X3 contributes with only one subunit to the heterotrimers while the other subunits could be P2X2, P2X4, or P2X5.

Ascorbylperoxide from parenteral nutrition induces an increase of redox potential of glutathione and loss of alveoli in newborn guinea pig lungs

BackgroundBronchopulmonary dysplasia is one of the main complications associated with extreme prematurity. Oxidative stress is suspected to be a trigger event of this lung disease, which is characterized by impaired alveolar development. Peroxides, mainly ascorbylperoxide and H2O2, are known contaminant of parenteral nutrition. We hypothesize that these oxidant molecules induce bronchopulmonary dysplasia development. The aim was to determine if the infusion of ascorbylperoxide, whether in presence or absence of H2O2, is associated with oxidative stress, apoptosis and loss of alveoli in the lungs of newborn guinea pigs. MethodThree-day-old guinea pigs received parenteral solutions containing 0, 20, 60 or 180 µM ascorbylperoxide in the presence or not of 350 µM H2O2 (concentrations similar to those measured in parenteral nutrition). After 4 days, the lungs were collected for determination of glutathione's redox potential, caspase-3 activation (an apoptosis marker), alveolarization index (by histology), activation of Nrf2 and NFκB (biological markers of oxidative stress), and IL-6 and PGJ2 levels (markers of NFκB activation). Groups were compared by ANOVA, p < 0.05. ResultsLoss of alveoli was associated with ascorbylperoxide in a dose-dependent manner, without an influence of H2O2. The dose-dependent activation of caspase-3 by ascorbylperoxide was lower in the presence of H2O2. Ascorbylperoxide induced an increase of redox potential in a dose-dependent manner, which reached a plateau in presence of H2O2. Nrf2 and NFκB were activated by H2O2 but not by ascorbylperoxide. ConclusionResults suggest that ascorbylperoxide, generated in parenteral nutrition, is involved in the development of bronchopulmonary dysplasia, independently of the increase of the redox potential. This study underlines the importance of developing a safer formulation of parenteral nutrition.

Maternal acrylamide treatment reduces ovarian follicle number in newborn guinea pig offspring

Acrylamide is an industrial chemical which has toxic effects on reproduction. In this study, we investigated whether acrylamide administered prenatally can induce follicular atresia in the newborn guinea pig ovary. Another aim was to describe the localization of vimentin filaments and determine their participation in atresia. After prenatal acrylamide treatment, the pool of primordial and primary follicles was significantly reduced. The number of caspase 3 and TUNEL positive oocytes increased compared to the control group. There were no differences in Lamp1 (autophagy marker) staining. A vimentin immunosignal was present in the granulosa cells of primordial, primary and secondary follicles. Interestingly, in contrast to the control group, the oocytes from all follicles in the ACR-treated females were negative for vimentin. These data suggest that prenatal exposure to acrylamide reduced the number of ovarian follicles by inducing follicular atresia mediated by oocyte apoptosis. Acrylamide-induced apoptosis may be associated with destruction of vimentin filaments.

Total parenteral nutrition induces sustained hypomethylation of DNA in newborn guinea pigs

Neonatal total parenteral nutrition (TPN) is associated with animals with low glucose tolerance, body weight, and physical activity at adulthood. The early life origin of adult metabolic perturbations suggests a reprogramming of metabolism following epigenetic modifications induced by a change in the pattern of DNA expression. We hypothesized that peroxides contaminating TPN inhibit the activity of DNA methyltransferase (DNMT), leading to a modified DNA methylation state. Three groups of 3-d-old guinea pigs with catheters in their jugular veins were compared: (i) control: enterally fed with regular chow; (ii) TPN: fed exclusively with TPN (dextrose, amino acids, lipids, multivitamins, contaminated with 350 ± 29 μmol/l peroxides); (iii) H2O2: control + 350 μmol/l H2O2 intravenously. After 4 d, infusions were stopped and animals enterally fed. Half the animals were killed immediately after treatments and half were killed 8 wk later (n = 4-6 per group) for hepatic determination of DNMT activities and of 5'-methyl-2'-deoxycytidine (5MedCyd) levels, a marker of DNA methylation. At 1 wk, DNMT and 5MedCyd were lower in the TPN and H2O2 groups as compared with controls. At 9 wk, DNMT remained lower in the TPN group, whereas 5MedCyd was lower in the TPN and H2O2 groups. Administration of TPN or H2O2 early in life in guinea pigs induces a sustained hypomethylation of DNA following inhibition of DNMT activity.

Morphological basis of lung injury by free radicals and the treatment in newborn guinea pigs

Summary. Structural and morphometrical lung changes in guinea pigs exposed to hyperoxia or induced reactive oxygen species (ROS) production during 14 days alone or under inhalations of N-acetylcysteine (in water solution or built-in multilayered liposomes), α-tocopherol or retinol have been studied. We established that induced ROS production in the lungs was associated with structural change of the lung tissue compatible with that in animals exposed to hyperoxia. Most typical changes associated with hyperoxia and induced ROS production were reduction in the alveolar intercept area and increase in the total area of the alveolar space and intrapulmonary vessel lumen. All the therapy regimens effectively prevented hyperoxiainduced changes of the alveolar space / interstitium relationship but were fail to prevent the dilation of the intrapulmonary vessels. The inhalation of N-acetylcysteine seemed to be the most effective treatment.