Untreated Pups Research Articles

Effects of maternal subclinical hypothyroidism on neurodevelopment of offspring- an animal-based study

Background and Objective: Maternal subclinical hypothyroidism (SCH) during pregnancy is associated with adverse maternal and neonatal outcomes, including cognitive and neuropsychiatric effects. This study aimed to assess the impact of maternal SCH on young rats, examining behavioral and gross brain structure changes, and evaluating reversibility after levothyroxine treatment. Methods: This experimental animal study was conducted at the Aga Khan University for a duration of 10 months. The study was approved by the Ethical committee for Animal Care and Use. Fourteen female Sprague Dawley rats were grouped into Treated (induced with SCH, treated with levothyroxine), Untreated (induced with SCH, no treatment), and Control (administered saline). Pups' body weight was monitored, and histological procedures were conducted at postnatal days 7, 14, and 21. Behavioral tests (elevated plus maze, forced swim, and tail suspension) assessed anxiety and depression. The data was analysed using SPSS (version 25.0). The mean, median, and standard deviation were calculated for each quantitative parameter. A one-way ANOVA was performed to identify any variation in mean thyroid levels between groups, and an independent sample t-test was utilized to confirm mean differences in anxiety levels. Results: On postnatal day 7, untreated pups showed lower body weight compared to treated and control groups. This trend continued on days 14 and 21. By day 21, an elevated plus maze (EPM) test indicated anxiety-like behaviour in untreated pups, while they also exhibited more signs of depression in tests like the forced swim test (FST) and tail suspension test (TST). Brain structure, including the prefrontal cortex, remained intact in offspring affected by maternal thyroid dysfunction, with no significant changes observed in brain morphology across the groups. Conclusion: Despite unchanged brain structure, untreated rat pups exhibited significant behavioral differences indicative of depression. This underscores the importance of understanding the behavioral impacts of maternal SCH even in the absence of gross anatomical alterations.

Induction of TNFα by flavin adenine dinucleotide (FADH) protects neonatal C57Bl6 lungs from high oxygen induced lung injury

Background: Bronchopulmonary dysplasia (BPD) is a serious lung disease caused by oxidative stress, predominately affecting premature infants that require prolonged oxygen support. The co-factor flavin adenine dinucleotide (FADH) facilitates glutathione reductase (GR) enzymatic activity and increases the bioavailability of the antioxidant glutathione (GSH). As such, we hypothesized that intranasal delivery of FADH can improve redox homeostasis and attenuate lung injury following chronic high oxygen supplementation (0.85% FiO2) by altering pro-inflammatory signal transduction pathways in a C57Bl6 newborn mouse model for BPD. Methods: We used a prolonged hyperoxia-induced lung injury rodent model to recapitulate the clinical and histological changes present in BPD. Newborn mice were housed in FiO2 85% (hyperoxic) or 21% O2 (normoxic) from day of birth until post-natal day (PN) 14. FADH (7 μM) or saline (control) treatments were administered daily via intranasal insufflation on PN 14-21 in a final volume of 1.5μL/g body weight. BALF and lung tissue samples were collected and evaluated for redox stress, lung injury/development, and inflammation using standard immunohistological techniques, morphometric analysis, and standard molecular assays. BALF cytokines were measured using a flow cytometric approach (BioLegend). Two-dimensional cell migration assays were tracked via microscopy daily until 100% confluence reached; the ½ maximal peak values were evaluated to compare rate of wound closure following very low (2.5 ng/mL) TNFα or IL12 treatment. Transepithelial resistance and potential differences were measured using an EVOM device (World Precision Instrument) across human small airway epithelial cells, and equivalent short circuit currents (Isc) calculated in accordance with Ohm’s Law, where V=IR. Single data comparisons were performed using paired Student’s t-tests. Multiple comparisons were performed using ANOVA followed by post-hoc testing in Sigma Plot. Results: FADH protected neonatal lungs from high oxygen induced oxidative stress: GSH/GSSG Eh (a measure of oxidative stress) improved from -168.77 mV ± 3.64 mV to -179.10 mV ± 1.85 mV (n=5 BALF measurements). FADH also improved lung injury scores from 0.187±0.038 to 0.03±0.014 (p=0.005), decreased neutrophil migration (p<0.001), and increased macrophages (p<0.001) when compared to age-matched untreated pups. FADH-mediated increase in TNFα (and not IL12) significantly increased the rate of lung epithelial cell wound closure from [2.27±0.07 to 1.97±0.1 average days for ½ complete wound closure, n=6, p=0.01] and increased Isc from 6.5±.21 to 9.2±1.0 μA/cm2, n=3, p=0.03. Conclusions: Our findings indicate that FADH protects the preterm lung from hyperoxic injury by 1) increasing the bioavailability of GSH and 2) via TNFα signaling to increase the rate of wound closure and repair of epithelial cell function. Significances: The mechanisms identified through this study could be targeted as a potential adjunct therapy for BPD patients to attenuate high oxygen induced lung injury. Funding: Supported by R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Surfactant Treatment Shows Higher Correlation Between Ventilator and EIT Tidal Volumes in an RDS Animal Model.

Neonatal respiratory distress syndrome (RDS) is a condition of pulmonary surfactant insufficiency in the premature newborn. As such, artificial pulmonary surfactant administration is a key treatment. Despite continued improvement in the clinical outcomes of RDS, there are currently no established bedside tools to monitor whether pulmonary surfactant is effectively instilled throughout the lungs. Electrical impedance tomography (EIT) is an emerging technique in which physiological functions are monitored on the basis of temporal changes in conductivity of different tissues in the body. In this preliminary study, we aimed to assess how EIT tidal volumes correlate with ventilator tidal volumes in an RDS animal model, namely untreated, surfactant-treated, and normal control rabbit pups. Tidal volumes were measured simultaneously on an EIT system and a mechanical ventilator and compared at different peak inspiratory pressures. The linear correlation between tidal volumes measured by EIT and by ventilator had an R 2 of 0.71, 0.76 and 0.86 in the untreated, surfactant-treated, and normal control groups, respectively. Bland–Altman analysis showed a good correlation between the measurements obtained with these two modalities. The intraclass correlation coefficients (ICC) between ventilator tidal volume and EIT tidal volume were 0.83, 0.87, and 0.93 (all p < 0.001) in the untreated, surfactant-treated, and normal control groups, respectively, indicating that the higher ICC value, the better inflated status of the lung. In conclusion, we demonstrated that EIT tidal volume correlated with ventilator tidal volume. ICC was higher in the surfactant treated group.

Lead exposure of rats during and after pregnancy induces anti-myelin proteolytic activity: a potential mechanism for lead-induced neurotoxicity

Toxic effects of lead (Pb) are principally manifested in the central nervous system (CNS) and a mounting body of evidence indicates that excessive chronic exposure to Pb participates in the pathological processes of numerous neurodegenerative disorders in humans.In this study we evaluated whether the prolonged pre- and postnatal exposure of rat pups to lead, administrated through ingestion in drinking water, as a typical environmental exposure, can determine alterations of the protein pattern of CNS myelin and the induction of myelin-associated proteinases. Pregnant dams were given distilled water or 0.3 mg/mL lead acetate in drinking water during gestation and lactation. At postnatal day (PND) 21, pups born from mothers poisoned with Pb continued the treatment with the metal. On PND 35 and 56, pups were sacrificed, and brains were subjected to myelin purification and extraction of myelin-associated proteinases. The SDS-PAGE analysis of protein pattern of myelin incubated in vitro with an oxidative system indicated that myelin proteins from Pb-treated pups were more sensitive to the toxicity of reactive oxygen species in comparison with those from untreated pups. The zymografic analysis of NaCl-extracts from myelin of Pb-treated pups showed a band of digestion of 54 kDa that increased in pups sacrificed at PND 56 in comparison with those sacrificed at PND 35 and correlated with the concentration of Pb, detected in purified myelin. The incubation of the NaCl-extract from Pb-treated pups with purified myelin basic protein (MBP) evidenced the presence of different MBP-degrading activities. These results suggest that Pb may influence the integrity of the myelin sheath, probably through the induction of anti-myelin proteinases.

Prenatal administration of heparin-binding epidermal growth factor-like growth factor in an experimental model of necrotizing enterocolitis decreased both incidence and severity of the disease.

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death in premature infants and causes long-term disabilities. Previously, enteral heparin-binding epidermal growth factor-like growth factor (HB-EGF) administered after birth demonstrated decreased incidence and severity of NEC in a neonatal animal model of NEC. We investigated the potential prophylactic strategy of preventing NEC using prenatally administered HB-EGF. An HB-EGF (800 µg/kg/dose) dose was injected into pregnant rats via tail vein or intraperitoneal route 2 hours prior to delivery. After cesarean section (C-section) at 21 days' gestation, the rat pups were subjected to the NEC protocol by inducing stressors: hypoxia, hypothermia, hypertonic feeds, and orogastric gavage of lipopolysaccharide (2 mg/kg). Postnatally, pups were monitored for 96 hours and assessed for the development of clinical and postmortem histological NEC. The experimental NEC incidence in untreated, stressed rat pups was 66%. Compared with untreated pups, the maternal administration of HB-EGF correlated with a significant NEC incidence and severity decrease in rat pups. The strongest decrease was seen when HB-EGF was administered via the intraperitoneal route 2 hours prior to C-section (66% vs 31%, *p<0.05). Prenatal HB-EGF administration significantly increased pups' survival after NEC protocol exposure, with the greatest benefit observed in the group that received HB-EGF intraperitoneally 2 hours before delivery. Prenatal administration of HB-EGF decreases the incidence and severity of NEC, preserves gut barrier function and increases survival. This may represent a novel prophylactic clinical strategy for NEC offered to mothers at risk of delivering a premature infant.

Topical ivermectin is a highly effective seal 'spot-on': A randomised trial of hookworm and lice treatment in the endangered Australian sea lion (Neophoca cinerea).

The Australian sea lion (Neophoca cinerea) is an endangered and declining otariid species, with a high rate of pup mortality associated with endemic hookworm (Uncinaria sanguinis) infection a suspected contributor to this decline. Injected ivermectin is an effective treatment for Uncinaria sp. in otariids, with optimal outcomes achieved by the early treatment of pups prior to disease development. This randomised controlled trial evaluated the effectiveness of the novel use of a topical ivermectin formulation against hookworm infection and lice (Antarctophthirus microchir) infestation, in comparison with injected ivermectin. During the 2017 breeding season at Dangerous Reef, South Australia, pups ≤ 70 cm in standard length (≤ 2 weeks of age; n = 85) were randomised to single dose topical (500 μg/kg spot-on; n = 27) or injected (200 μg/kg subcutaneous; n = 29) ivermectin treatment groups, or to an untreated control group (n = 29). Topical ivermectin was highly effective for U. sanguinis elimination, and not significantly different to the injected formulation (estimated effectiveness 96.4% and 96.8%, respectively; P > 0.05). Its application resulted in an 81.6% reduction and 62.7% additional clearance for A. microchir infestation by 15–24 days post-treatment, compared with untreated control pups (also not significantly different to injected ivermectin; 83.1% and 59.4%, respectively; P > 0.05). Treatment with either ivermectin formulation significantly ameliorated increases in inflammatory markers detected in the blood of untreated control pups – peripheral blood eosinophil counts (persisting to 36–41 days post-recruitment P < 0.05) and increased plasma protein concentrations (15–24 days post-recruitment; P < 0.05). Further, an initial short-term decrease in body condition in the control group was not observed in either of the treatment groups. This study demonstrates that topical ivermectin is an effective antiparasitic treatment in N. cinerea. It offers an alternative administration method for ivermectin delivery to a young pup cohort in this species, and an alternative, minimally invasive management tool for species conservation.

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in sphingosine-1-phosphate lyase 1 (SGPL1), which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first 2 years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9–mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1-KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated proinflammatory and profibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared with untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis, and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.

Phenobarbital and midazolam suppress neonatal seizures in a noninvasive rat model of birth asphyxia, whereas bumetanide is ineffective.

Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in ~50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. Intermittent asphyxia was induced for 30minutes by exposing the rat pups to three 7+3-minute cycles of 9% and 5% O2 at constant 20% CO2 . Drug treatments were administered intraperitoneally either before or immediately after asphyxia. All untreated rat pups had seizures within 10minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30mg/kg but not 15mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3mg/kg) alone or in combination with phenobarbital (15 or 30mg/kg) exerted no significant effect on seizure occurrence. The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.

MEKK3 Regulates Angiogenesis through Smad Signaling

Angiogenesis is one of the most essential events during embryonic development. The previous studies proved that MEKK3 (MAP3K3) is critical for angiogenesis. However, precisely how MEKK3 regulates the vascular development remains largely unknown. To investigate the impact of MEKK3 in vascular development in vivo and its role in normal physiological and pathological settings, we generated VE-Cadherin CreERT2;MEKK3 flox/+ (control) and VE-Cadherin CreERT2;MEKK3 flox/- (MEKK3 iEC -/-) mice. MEKK3 deletion was induced from postnatal day (P) 1 by administration of tamoxifen daily. The retinal angiogenesis model was used to analyze the blood vessel development in P7 and P12 mice. Whole-mount staining of P7 retinas in MEKK3 iEC -/- mutants showed a significant decrease in vascularized area and branch formation. In contrast, control pups, although only having one copy of the MEKK3 gene in endothelial cells, developed a complete vascular sheet at the same dose of tamoxifen, as did the untreated pups. At P12, MEKK3 iEC -/- mice showed defects in vessel remodeling and arterial patterning. These retinas had dilated and tortuous arteries losing the identical marker. This phenotype accorded with the pathology of AVM (arteriovenous malformation). We then cultured murine aortic rings in 3D matrigel and induced sprouting with VEGF. Administration of TGF-b receptor kinase inhibitor SB431542 to the MEKK3 iEC-/- aorta showed a partial rescue effect on the number of sprouts and vascularized area. It implicated that the vascular malformation in the MEKK3 iEC-/- mice was partially due to the elevated TGF-b signals in endothelial cells. We confirmed the result by staining the retinas with SMA and N-cadherin, which are the downstream genes in TGF-b pathway. It turned out that both SMA and N-cadherin were expressed stronger in MEKK3 iEC-/- retinas than in the control ones. Next, by in vitro studies, we found that endothelial cell activation by VEGF treatment led to rapid and strong phosphorylation of R-SMADs at the linker regions in the control endothelial cells. Such phosphorylation was severely impaired in the MEKK3 -/- endothelial cells. The same results were found in the activation of ERK1/2 and p38. Inhibition of ERK1/2 but not p38 MAPK in control endothelial cells greatly reduced the VEGF-induced phosphorylation of R-SMADs at their linker regions. Inhibition of both ERK1/2 and p38 did not further reduce the SMAD linker region phosphorylation compared to inhibition of ERK1/2. Thus, we determined ERK1/2 are responsible for the VEGF-induced, MEKK3-dependent phosphorylation of R-SMADs at the linker region. Finally, since Notch pathway is an evolutionarily conserved signaling , which directly affects angiogenesis, we used aortic ring model to test their interaction, and detected the gene expression in endothelial cells. Hey1 and Hey2 were over expressed in MEKK3 -/- endothelial cells. Knockdown of Hey1 and Hey2 could rescue the deficient angiogenesis. Knockdown of RBPJ did not affect MEKK3 mediated angiogenesis or induction of Hey1 and Hey2. The above data supported a model whereby MEKK3-mediated SMAD signaling induces HEY1 and HEY2 independently of canonical Notch activation. Disclosures No relevant conflicts of interest to declare.

Caffeine Restores Background EEG Activity Independent of Infarct Reduction after Neonatal Hypoxic Ischemic Brain Injury

In human preterm newborns, caffeine increases brain activity and improves neurodevelopmental outcomes. In animal models of hypoxic ischemic brain injury, caffeine pretreatment reduces infarct volume. We studied the relationship between tissue neuroprotection and brain activity after injury to further understand caffeine neuroprotection. Rat dams received caffeine prior to birth or on postnatal day 3 (P3) through P16. Caffeine-treated and -untreated pups underwent the Vannucci procedure (unilateral carotid ligation, global hypoxia) on P2. A subset had EEG recordings. Brain hemispheric infarct volume was measured on P16. P2 hypoxic ischemia (HI) results in histologic brain injury (mean ± standard deviation infarct volume 10.3 ± 4.6%) and transient suppression of EEG activity. Caffeine pretreatment reduces brain injury (mean ± standard deviation infarct volume 1.6 ± 4.5%, p < 0.001) and improves amplitude-integrated EEG (aEEG) and EEG burst duration and amplitude. Caffeine treatment after HI does not reduce infarct volume (mean ± standard deviation 8.3 ± 4.1%, p = 1.0). However, caffeine posttreatment was equally effective at restoring aEEG amplitude and EEG burst duration and amplitude. Thus, caffeine supports brain background electrical activity independent of tissue neuroprotection.

A103 PROBIOTIC FORMULATIONS DIFFERENTIALLY AFFECT DISEASE OUTCOME IN A MOUSE MODEL OF NECROTIZING ENTEROCOLITIS

Abstract Background Necrotizing enterocolitis (NEC) causes severe gastrointestinal disease in premature infants. A precise etiology is not yet known, but it is generally agreed that intestinal immaturity &amp; alterations in the gut microbiota play key roles in NEC progression. There is growing interest in the clinical use of probiotics for the prevention of NEC in at-risk infants. Some probiotics, which are defined as beneficial bioactive microbes that provide a health benefit when administered in sufficient amounts, may influence disease outcome in premature infants with NEC. Aims To investigate the efficacy of two commercially available probiotic formulations, Lacidofil® and ProBiokid®, in the prevention of experimentally-induced NEC. Methods At gestational day 12.5, treated dams were administered probiotics (109 cfu/mL) in drinking water (Table 1). NEC was induced at postnatal day 5 or when pups were ≥ 3.0 g by the administration of hyperosmolar formula, hypoxia, and lipopolysaccharide (4 mg/kg). NEC pups receiving probiotics were administered 108cfu in hyperosmolar formula once daily during the morning feeding. Body weight, survival and disease severity were scored. Results Neither probiotic intervention affected overall survival of NEC-induced pups. Control dam-fed pups (NN) continued to gain weight as expected over time, while untreated NEC pups (NNNEC) exhibited a significant delay in weight gain. LLNEC pups exhibited the greatest percent body weight increase over time, possibly due to a lower birth weight. Lacidofil administration did not improve NEC-induced tissue damage irrespective of the timing of intervention. In contrast, Probiokid provided to both dam and pups (PPNEC) resulted in less severe tissue damage compared to their control counterparts. Conclusions These finding demonstrate strain-specific differences in two different commercial probiotic formulations in the prevention of NEC in newborn mice. Future studies seek to provide insight into how Lacidofil and Probiokid engage intestinal stem cells (Lgr5-GFP mice), cell proliferation (Ki67 positivity) and differentiation (MUC2 &amp; Chromogranin A positivity) in the NEC-damaged intestine. Funding Agencies CIHRMITACS Accelerate Postdoctoral Award

Risk Assessment of Cosmetics Using Triclosan on Future Generation's Germ Cell Maturation via Lactating Mother Rats.

Triclosan (TCS) is a widely used chemical in personal care and household products as an antimicrobial agent but some studies have reported it as being estrogenic. We investigated the influence of TCS on the male reproductive system of postnatal pups. Lactating mother rats (Rattus norvegicus) were given daily doses of 0 mg, 3 mg, and 5 mg/kg/day from the day of delivery until 28 days, equivalent to their natural breastfeeding duration. At 28 days, the male pups of all three groups were sacrificed and their biochemical parameters evaluated. TCS-treated pups had decreased mRNA levels for 3β hydro-hydroxysteroid dehydrogenases (3βHSD), OCT3/4, and androgen receptor (AR) (p < 0.05). The higher dose (5 mg/kg/day) male pups exhibited more significantly affected germ cell maturation and decreased body weight. In summary, TCS-treated lactating mothers passed the deleterious effects to their untreated male pups as exhibited by reduced androgens synthesis and subsequently decreased sperm count.

Paternal cadmium exposure increases the susceptibility to diet-induced testicular injury and spermatogenic disorders in mouse offspring.

The impairments of gestational cadmium (Cd) exposure on testicular development and male fertility in offspring have been reported. Here, we investigated the effect of paternal low-concentration cadmium exposure on testicular development and spermatogenesis in offspring. Five-week-old male mice were exposed to cadmium chloride (100mg/L) in drinking water for 20 weeks. Results presented that Cd did not affect the testicular histology and sperm count in mice. After mating with untreated females, pregnant mice and pups were then evaluated. No significant difference in the rate for successful pregnancy and the body weight of pups was observed in Cd-exposed mice compared to the controls. Male offspring were given with a chow and high-fat diet from postnatal day (PND) 35 to PND70. Our data indicated that high-fat diet obviously decreased No. of sperm in epididymides of adult offspring due to paternal Cd exposure. Testicular histology revealed that the percentage of seminiferous tubules in stages IX-XII and the atypical residual bodies positive tubules in CdH (paternal cadmium exposure and pubertal high-fat diet) group were higher than these in CdC (paternal cadmium exposure and pubertal chow diet) group. Further analysis demonstrated that high-fat diet markedly accelerated testicular apoptosis, as determined by TUNEL assay and immunostaining for cleaved caspase-3, in male offspring due to paternal Cd exposure. Collectively, high-fat diet exacerbates the damage of testicular development and spermatogenesis in offspring due to paternal cadmium exposure.

Retinoic acid: A potential therapeutic agent for cryptorchidism infertility based on investigation of flutamide-induced cryptorchid rats in vivo and in vitro

Cryptorchidism is a common disorder in children and may cause infertility in adults. The BTB is essential for maintaining the microenvironment necessary for normal spermatogenesis. This study investigated whether retinoic acid (RA) may regulate the proteins that are essential for integrity of the BTB in cryptorchidism. Female Sprague-Dawley rats were administrated flutamide during late pregnancy to induce a model of cryptorchidism in male offspring. The concentrations of RA and BTB tight and gap junction protein levels were significantly lower in untreated cryptorchid pups compared with normal pups, but almost normal in cryptorchid pups given RA. Studies in vitro corroborated these findings. The sperm quality of RA-treated model pups was better compared with the untreated model. RA treatment may have therapeutic potential to restore retinoic acid and proteins associated with integrity of the BTB in cryptorchid testis.

Galactagogue Effect of &amp;lt;i&amp;gt;Euphorbia hirta&amp;lt;/i&amp;gt; (Euphorbiaceae) Aqueous Leaf Extract on Milk Production in Female Wistar Rats

Background: Populations of Gontougo (Côte d’Ivoire) commonly used Euphorbia hirta (Euphorbiaceae) leaves as galactagogue to boost milk production in lactating women. Our study was carried to verify this traditional belief via evaluations of milk production, serum prolactin levels, body weight and mammary lobuloalveolar proliferation induced by the aqueous leaf extract of this medicinal plant in Wistar rats. Methods: 6 groups of 6 virgin rats and 6 groups of 6 lactating rats were used. Each lactating rat was isolated with its six pups. These groups of animals were treated orally with distilled water, Metoclopramide (Metocl 5 mg/kg) and E. hirta aqueous leaf extract (EHae 100, 200, 400 and 600 mg/kg) daily during 17 days of lactation. The indirect method which assimilates the weight gain of untreated pups to the quantity of milk produced by lactating rats was used. At the end of the treatment, histological sections of the abdominal mammary glands of lactating rats were observed. Results: EHae administration induced an increase of milk production in lactating rats. This action of EHae was stronger at the dose of 200 mg/kg. Milk production of the treated rats at a dose of 200 mg/kg was 118.14 ± 1 g. The quantity of milk produced by the control female rats was 83.83 ± 0.88 g. Compared with the control, EHae 200 mg/kg significantly increased milk production (40.92%) (P < 0.0001). EHae 200 mg/kg significantly increased the biosynthesis of prolactin (PRL) in virgin rats (P < 0.0001). At this dose, PRL values measured at D7 and D14 were 26.44 ± 8.64 and 33.88 ± 7.30 ng/ml respectively, whereas the value obtained in the controls that received distilled water was less than 0.5 ng/ml. Histological analysis showed that EHae 200 mg/kg induced the development of the lobuloalveolar system of the mammary glands. Conclusion: EHae showed a galactagogue activity illustrated by increases of milk production, PRL secretions and the development of the lobuloalveolar system of the mammary glands in treated female rats. This galactagogue property could partly justify the traditional use of this plant to boost milk production in lactating women.

Perinatal and long-term effects of maternal uterine artery adenoviral VEGF-A165 gene therapy in the growth-restricted guinea pig fetus.

Uterine artery application of adenoviral vascular endothelial growth factor A165 (Ad.VEGF-A165) gene therapy increases uterine blood flow and fetal growth in experimental animals with fetal growth restriction (FGR). Whether Ad.VEGF-A165 reduces lifelong cardiovascular disease risk imposed by FGR remains unknown. Here, pregnant guinea pigs fed 70% normal food intake to induce FGR received Ad.VEGF-A165 (1×1010 viral particles, n = 15) or vehicle ( n = 10), delivered to the external surface of the uterine arteries, in midpregnancy. Ad libitum-fed controls received vehicle only ( n = 14). Litter size, gestation length, and perinatal mortality were similar in control, untreated FGR, and FGR+Ad.VEGF-A165 animals. When compared with controls, birth weight was lower in male but higher in female pups following maternal nutrient restriction, whereas both male and female FGR+Ad.VEGF-A165 pups were heavier than untreated FGR pups ( P < 0.05, ANOVA). Postnatal weight gain was 10-20% greater in female FGR+Ad.VEGF-A165 than in untreated FGR pups, depending on age, although neither group differed from controls. Maternal nutrient restriction reduced heart weight in adult female offspring irrespective of Ad.VEGF-A165 treatment but did not alter ventricular wall thickness. In males, postnatal weight gain and heart morphology were not affected by maternal treatment. Neither systolic, diastolic, mean arterial pressure, adrenal weight, nor basal or challenged plasma cortisol were affected by maternal undernutrition or Ad.VEGF-A165 in either sex. Therefore, increased fetal growth conferred by maternal uterine artery Ad.VEGF-A165 is sustained postnatally in FGR female guinea pigs. In this study, we did not find evidence for an effect of maternal nutrient restriction or Ad.VEGF-A165 therapy on adult offspring blood pressure.

Treatment of experimental necrotizing enterocolitis with stem cell-derived exosomes

PurposeNecrotizing enterocolitis (NEC) remains a devastating disease in premature infants. We previously showed that four stem cell (SC) types equivalently improve experimental NEC. Exosomes are intercellular nanovesicles containing RNA, miRNA, DNA, and protein. Because SC therapy faces challenges, our aim was to determine if the beneficial effects of SC are achievable with cell-free exosomes. MethodsExosomes from four SC types were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) bone marrow-derived MSC (BM-MSC); (3) amniotic fluid-derived neural SC (AF-NSC); and (4) neonatal enteric NSC (E-NSC). Rat pups exposed to NEC received a varying concentration of a single type of exosome with control pups receiving PBS only. Intestinal damage was graded histologically. ResultsThe incidence of NEC was 0% in unstressed pups and 60.7% in control pups subjected to NEC. Exosomes (4.0×108) reduced NEC incidence to: AF-MSC 25.0%; BM-MSC 23.1%; AF-NSC 11.1%; E-NSC 27.3%. When administered at a concentration of at least 4.0×108, all groups demonstrated a significant reduction in NEC compared to untreated pups. At this minimum concentration, there was no difference in treatment efficacy between exosomes and the SC from which they were derived. ConclusionStem cell-derived exosomes reduce the incidence and severity of experimental NEC as effectively as the stem cells from which they are derived, supporting the potential for novel cell-free exosome therapy for NEC. Type of studyBasic science.

Laminin-521 Protein Therapy for Glomerular Basement Membrane and Podocyte Abnormalities in a Model of Pierson Syndrome.

Background Laminin α5β2γ1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity.Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2-/- pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture.Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM.Conclusions These studies show that GBM composition and function can be altered in vivovia vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired.

FLLL32, a curcumin analog, ameliorates intestinal injury in necrotizing enterocolitis.

BackgroundNecrotizing enterocolitis (NEC) is a devastating gastrointestinal disease that primarily affects premature infants. It is characterized by inflammation and leukocyte infiltration that can progress to intestinal necrosis, perforation, systemic inflammatory response, and death. In this study, we examined the effect of FLLL32, a curcumin analog, on an NEC-like neonatal intestinal injury model.MethodsNEC was induced in CD-1 mice pups using the Paneth cell ablation and Klebsiella infection model. Pups were divided into sham, NEC, and NEC + FLLL32 groups. At the end of the experiment, pups were euthanized; whole blood and small intestines were harvested. Intestinal inflammatory cytokine profile, in vivo intestinal permeability using serum fluorescein isothiocyanate-dextran, and histological injury scores were compared between the groups.Results and conclusionFLLL32-treated pups had lower intestinal injury, improved intestinal permeability, and lower proinflammatory cytokine profiles compared to those in untreated pups with NEC. These results suggest that FLLL32 plays a protective role in NEC.

Evaluating the efficacy of different types of stem cells in preserving gut barrier function in necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. Increased intestinal permeability is central to NEC development. We have shown that stem cells (SCs) can reduce the incidence and severity of NEC. Our current goal was to investigate the efficacy of four different types of SC in preservation of gut barrier function during NEC. Materials and methodsWe compared (1) amniotic fluid–derived mesenchymal SC, (2) bone marrow–derived mesenchymal SC, (3) amniotic fluid–derived neural SC, and (4) enteric neural SC. Premature rat pups received an intraperitoneal injection of 2 × 106 SC or phosphate-buffered saline only and were then subjected to experimental NEC. Control pups were breastfed and not subjected to NEC. After 48 h, animals received a single enteral dose of fluorescein isothiocyanate -labeled dextran (FD70), were sacrificed 4 h later, and serum FD70 concentrations determined. ResultsCompared to breastfed, unstressed pups with intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 μg/mL), untreated pups exposed to NEC had impaired barrier function with significantly increased permeability (18.6 ± 4.25 μg/mL, P = 0.047). Pups exposed to NEC but treated with SC had significantly reduced intestinal permeability: Amniotic fluid–derived mesenchymal SC (9.45 ± 1.36 μg/mL, P = 0.017), bone marrow–derived mesenchymal SC (6.73 ± 2.74 μg/mL, P = 0.049), amniotic fluid–derived neural SC (8.052 ± 1.31 μg/mL, P = 0.0496), and enteric neural SC (6.60 ± 1.46 μg/mL, P = 0.033). ConclusionsSCs improve gut barrier function in experimental NEC. Although all four types of SC reduce permeability equivalently, SC derived from amniotic fluid may be preferable due to availability at delivery and ease of culture, potentially enhancing clinical translation.