Birth In Rats Research Articles

Ultrastructural Features of Left Ventricle Cardiomyocytes in Preterm Newborn Rats.

The structure of left ventricular cardiomyocytes of 1 day preterm newborn rats was studied using transmission electron microscopy. It was shown that the relative area of the nucleus in cardiomyocytes of preterm rats is lower, and the relative area of the cytoplasm is higher than in full-term rats, while the relative areas of myofibrils and mitochondria do not differ. In cardiomyocytes of preterm rats damaged mitochondria, subsegmental myofibrillar contracture, and cytoplasmic swelling were found on the first postnatal day. Preterm birth in rats, in contrast to birth at term, is accompanied by the development of a number of ultrastructural damages in cardiomyocytes.

Functionalized human umbilical cord mesenchymal stem cells and injectable HA/Gel hydrogel synergy in endometrial repair and fertility recovery

Intrauterine adhesions (IUA) caused by endometrial injury are one of the main causes of female infertility. The current treatments for endometrial injury offer limited clinical benefits and cannot improve endometrial receptivity and pregnancy outcomes. Tissue engineering and regenerative medicine are considered potential solutions to address this concern and may offer effective treatment methods for the regeneration of injured human endometrium. Herein, we prepared an injectable hydrogel based on oxidized hyaluronic acid (HA-CHO) and hydrazide-grafted gelatin (Gel-ADH). The injectable hydrogel showed satisfactory biocompatibility when mixed with human umbilical cord mesenchymal stem cells (hUCMSCs). In an endometrial injury rat model, the treatment with hUCMSCs-loaded injectable hydrogel significantly enhanced the thickness of the endometrium and increased the abundance of blood vessels and glands in the injured endometrium compared to the control group. The hUCMSCs-loaded injectable hydrogel treatment significantly reduced endometrial fibrosis, decreased the expression of the pro-inflammatory factors (IL-1β and IL-6) and increased the expression of the anti-inflammatory factor (IL-10). This treatment induced endometrial VEGF expression by activating the MEK/ERK1/2 signaling pathway. Moreover, this treatment improved endometrial receptivity to the embryo and restored the embryo implantation rate similar to the sham group (48% in the sham group vs 46% in the treatment group), and this treatment achieved pregnancy and live birth in rats with endometrial injury. In addition, we also preliminarily validated the safety of this treatment in the maternal rats and fetuses. Collectively, our study showed that the hUCMSCs-loaded injectable hydrogel hold potential as an effective treatment strategy promoting rapid recovery of endometrial injury, and this hydrogel is a promising biomaterial for regenerative medicine applications. Statement of significance1. Oxidized hyaluronic acid (HA-CHO)/hydrazide-grafted gelatin (Gel-ADH) hydrogel combined with human umbilical cord mesenchymal stem cells (hUCMSCs) are effective in improving the regeneration of endometrium in the endometrial injury rat model.2. The hUCMSCs-loaded hydrogel treatment promotes the expression of endometrial VEGF through MEK/ERK1/2 signaling pathway and regulates the balance of inflammatory factors.3. The embryo implantation and live birth rates restore to normal level in the endometrial injury rat model, and the hydrogel has no adverse effects on maternal rats, fetuses, and offspring development after the treatments.

T-cells regulate albuminuria but not blood pressure or tissue damage in the Dahl SSCD247+/+ rat model of hypertension

Previous studies have demonstrated a key role for renal inflammation in the progression of salt-sensitive hypertension (SS-HTN). Pharmacological and genetic inhibition of inflammation blunts the progression of hypertension in several models, including Dahl salt-sensitive (SS) rats. Traditionally, it has been difficult to separate the pathological effects of renal T-cells from those of exposure of the kidney to elevated pressure since the inhibition of renal inflammation results in a reduction in blood pressure, and vice versa. Therefore, improved renal function and reduced injury after the suppression of inflammation may be the result of reduced renal T-cells, reduced blood pressure, or both. In these studies, we took advantage of a loss of protection from SS-HTN in SS rats lacking T-cells, SS CD247-/- rats, to isolate the pathological effects of renal T-cells from those of hypertension on the kidney.Studies were performed on male SS CD247+/+ and SS CD247-/- rats obtained from heterozygous breeders. From birth rats were maintained on a purified control salt diet (CS, 0.4% NaCl) and switched to a high-salt diet (HS, 4.0% NaCl) during experiments.In contrast to previous studies, we found that MAP following 21-days of HS was not significantly different between SS CD247+/+ and SS CD247-/- rats (SS CD247+/+ : 156 ± 2.2 mmHg, n=16 vs SS CD247-/- : 151 ± 2.2mmHg, n=14, p=0.05). Using flow-cytometry we confirmed that the loss of protection from SS-HTN was not due to the reconstitution of T-cells in the SS CD247-/- rats. T-cells were absent in the kidneys of SS CD247-/- rats after 21-days of HS, comprising 0.06 ± 0.015% of live renal cells, compared to 26.08 ± 2.13% in SS CD247+/+ rats (p= 0.006, n=4+7).Despite the equivalent levels of SS-HTN, we found that the SS CD247-/- rats demonstrated significantly lower levels of albuminuria than their wild-type littermates (528 ± 73 mg/g, n=5 vs 963 ± 156 mg/g, n=7, p=0.008). In contrast to the albuminuria, we found that kidney injury, specifically tubular protein casts and glomerular injury, were not significantly different between SS CD247+/+ and SS CD247-/- rats, while there was a significant correlation between these factors and MAP at tissue collection. RNA-sequencing data confirmed this correlation between injury and MAP – only 99 genes differed significantly in the outer medulla of SS CD247-/- and SS CD247+/+ rats, while >4000 genes differed significantly across the range of pressures evaluated.These findings demonstrate that the pathological effects of renal T-cells are primarily manifested in albuminuria and that the effects of pressure substantially outweigh the effects of T-cell infiltration on histologic injury and transcriptomics of the kidney in SS-HTN. R01HL152166. This is the full abstract presented at the American Physiology Summit 2023 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.

Effect of preterm birth in rats on proliferation and hyperplasia of cardiomyocytes

Aim. To identify the effect of preterm birth on proliferation and hyperplasia of cardiomyocytes in the early postnatal period of ontogenesis in rats.Materials and methods. Preterm birth (on day 21 and 21.5 of gestation) in Wistar rats was induced by subcutaneous administration of mifepristone. Immunohistochemistry was used to identify and calculate the number of Ki67-positive and Mklp2-positive cardiomyocytes in the left ventricle of preterm and full-term rats on days 1, 2, 3, 4, 5, and 6 of postnatal ontogenesis. Statistical analysis of morphometric parameters was performed using the Shapiro – Wilk test and Mann – Whitney test with the Bonferroni correction.Results. We revealed an increase in the number of Ki67-positive cardiomyocytes in the left ventricle of the rats: on day 1 of postnatal ontogenesis (in the rats born on day 21 of gestation) and on days 3–5 of postnatal ontogenesis (in the rats born on day 21.5 of gestation). Preterm birth in rats did not result in a change in the number of Mklp2-positive cardiomyocytes in the left ventricular wall.Conclusion. A change in the pattern of Ki67 expression by cardiomyocytes in the rats born 12 or 24 hours before full term was demonstrated in the early postnatal period of ontogenesis. An isolated increase in Ki67 expression without a change in Mklp2 expression by cardiomyocytes in the left ventricular wall of preterm rats indicates acceleration of cardiomyocyte hypertrophy. Shorter duration of prenatal development is associated with more pronounced morphological and functional rearrangements in the rat myocardium.

A5 noradrenergic neurons and breathing control in neonate rats.

The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DβH-SAP) to specifically ablate noradrenergic neurons. Thus, DβH-SAP (420ng/μL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).

MORPHOLOGICAL FEATURES OF RATS’ HEARTS AFTER INTRAFETAL INJECTION OF DEXAMETHASONE

The aim: Is to study the morphological features of rats' hearts after prenatal administration of glucocorticoids. Materials and methods: In this study we used histological, immunohistochemical, electron-microscopic and statistical research methods. Results: It is found that at 30th day after birth in rats after intrafetal introduction of dexamethasone in myocardium a relative area occupied by arterial vessels is significantly smaller in comparison with control. Absolute and relative number of Ki-67+-cardiomyocytes in the myocardium of experimental rats is reduced throughout the second week after birth and is significantly less compared to the control group. In the nuclei of cardiomyocytes of experimental rats is rendered the greater amount of heterochromatin in comparison with cardiomyocytes of the control group where euchromatin prevails. Conclusions: After intrafetal injection of dexametazone changes in dynamics and significantly smaller index of relative area occupied by arterial vessels in ventricular myocardium at the 30th day after birth are observed; the absolute and relative number of Ki-67+ -cardiomyocytes in myocardium decreases during the second week after birth and is significantly lower compared to the control group; in the nuclei of cardiomyocytes of experimental rats a greater amount of heterochromatin is visualized, and in cardiomyocytes of the control group - euchromatin.

Immunohistochemical analysis of protective effects of maternal fingolimod on the placenta and fetal lung and brain in chorioamnionitis-induced preterm birth rat model

Objectives Fingolimod (FIN) is used for multiple sclerosis treatment and has potential antiapoptotic and anti-inflammatory effects. We aimed at expanding our knowledge on various immunohistochemical markers for elucidating the possible mechanisms of action of fingolimod in the placenta and fetal lung and brain. Methods Sixteen pregnant rats were divided into four groups. On gestational day 17, lipopolysaccharide (LPS) was injected intraperitoneally to induce preterm fetal injury followed by intraperitoneal injection of fingolimod. Hysterotomy for preterm delivery was performed 6 h after fingolimod was injected. The study groups included (1) control, (2) LPS (1 mg/kg), (3) FIN (4 mg/kg), and (4) FIN + LPS. Fetal brain and lung and placenta samples were collected for histopathological examination. Moreover, fetal lungs (surfactant protein-A (SP-A), SP-B, SP-D, caspase-3, and caspase-8), fetal brains (interleukin-10, interleukin-1β, TNF-α, caspase-8, glial fibrillary acidic protein, vimentin, myelin basic protein, and receptor activator of nuclear factor kappa), and placenta tissues (interleukin-10, interleukin-1β, TNF-α, caspase-3, and caspase-8) were immunohistochemically evaluated. Results Maternal fingolimod treatment led to attenuation of LPS-induced fetal brain, lung, and placental injury, as indicated by lower immunoexpression of inflammatory markers compared to LPS group (p < .0001 for all comparisons). Conclusion The findings of the present study confirm the neuroprotective effects of antenatally administered fingolimod, which also significantly improved preterm fetal lung injury and placental inflammation in LPS-exposed preterm pregnancies by possible antiapoptotic and anti-inflammatory effects.

Primary study on the mechanism of ductus arteriosus closure in rats: impact of aging pathway

Objective: To explore the role and mechanism of aging pathway in patent ductus arteriosus closure of rats. Methods: Thirty outbreeding Sprague Dawley rats(20 females, 10-15 weeks old, 270-330 g) underwent random mating and conception. The primary Ductus Arteriosus smooth muscle cells (DASMCs) of pregnant 19 days(E19 group), 21 days(E21 group) and newborn(Day0 group) fetus were extracted and cultured. mRNA expression of cell senescence related markers p16, 21 and 53 genes in each group were detected by real-time fluorescent quantitative PCR(RT-PCR) after 48 hours culture. After hypoxic culture on DASMCs for 3 days, the DASMCs were divided into 3 groups: hypoxic control group(G0 group), 3 hours normal oxygen concentration treatment group(G3 group) and 6 hours normal oxygen concentration treatment group(G6 group). After intervention, mRNA expression of p16, 21 and 53 RT-PCR was detected. The DASMCs of newborn rats(Day0 group) were extracted and divided into 3 groups:low-oxygen culture control group, low-oxygen+siRNA culture group and normal oxygen concentration culture group. The DASMCs migration ability was tested experimentally by Transwell method. Result: The mRNA levels of p16, 21 and 53 in DASMCs were higher in E19 group than in Day0 group(all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were also higher in E21 group than those in Day0 group (all P<0.01). The mRNA levels of p16, 21 and 53 in DASMC were all higher in G0 group than those in G3 group (P<0.05 or 0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G0 group than those in G6 group (all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G3 group than those in G6 group (all P<0.05). DASMCs migration ability of newborn rats was higher in normal oxygen concentration culture group than that in low-oxygen culture group (P<0.01), and DASMCs migration ability of newborn rats was also higher in low-oxygen+siRNA culture group than that in low-oxygen culture group (P<0.01). Conclusion: The expression of senescence marker of DASMCs decreases with the birth in rats during the process of ductal closure, and the aging pathway may affect ductal closure by inhibiting DASMCs migration in rats.

Postnatal development in the rat: Changes in Na+ flux, sodium pump molecular activity and membrane lipid composition

The cellular mechanisms underpinning changes in metabolism during postnatal development in young mammals have not been extensively examined. This study examines changes in sodium pump capacity (Na+, K+-ATPase activity), number and molecular activity, as well as, Na+ flux, cholesterol level and fatty acid composition in a number of major organs during postnatal development in the rat. In liver, Na+ flux was highest (2.6 times) in the youngest rats (3-day old) and decreased with increasing age, whereas Na+, K+-ATPase activity increased with age (up to 9–28 days) in liver, kidney and brain, but not in heart. Increases in Na+, K+-ATPase activity where primarily driven by increases in molecular activity, 4-fold in brain and 7-fold in kidney, rather than by increases in sodium pump number. Membrane polyunsaturation increased in both kidney and brain during development, with kidney becoming increasingly dominated by omega-6 (18:2n-6 and 20:4n-6) and brain by omega-3 (22:6n-3) fatty acids. Membrane reconstitution experiments support the concept that changes in membrane composition might underpin higher sodium molecular activities in the adult. In conclusion, at birth rats possess high Na+ flux but a lower sodium pump capacity that increases with age being driven by increases in molecular activities associate with changes in membrane lipid composition.

Effect of low- and high-protein maternal diets during gestation on reproductive outcomes in the rat: a systematic review and meta-analysis.

Studies with animal models have consistently demonstrated adverse health outcomes in offspring born following nutritional manipulation during gestation. However, the effects of gestational dietary protein modification on reproductive outcomes at birth are less clear. We, therefore, conducted a systematic review and meta-analysis of controlled trials to determine whether high- or low-protein diets are associated with altered reproductive outcomes in a commonly studied species, the rat. Included studies were identified through a systematic search using electronic databases and manual literature review to identify randomized studies published between June 1972 and March 2019. Thirty-two studies were identified and used to analyze the effects of low- and high-protein gestational diets on litter size, litter weight, gestational weight gain, and gestational feed intake. The results indicate that low-protein diets significantly reduced litter weight (P < 0.00001) and gestational weight gain (P < 0.0006), but did not influence litter size (P = 0.62) or gestational feed intake (P = 0.25). In contrast, high-protein diets were found to reduce gestational feed intake (P = 0.004) but did not influence litter size (P = 0.56), litter weight (P = 0.22), or gestational weight gain (P = 0.35). The results suggest that low but not high-protein gestational diets alter reproductive outcomes at birth in rats.

Prepubertal PTU treatment in rat increases Sertoli cell number and sperm production.

The number of Sertoli cells (SCs) ultimately determines the upper limit of sperm production in the testis. Previous studies have shown that thyroid hormones (TH) receptors are abundantly expressed in developing SCs; therefore, it was highly significant to discover that transient neonatal hypothyroidism induced by the goitrogen 6-n-propyl-2-thiouracil (PTU) can extend SCs proliferation beyond the first 2 weeks postnatal and increase testis weight and sperm production. Further studies concluded that treatment must begin before day 8 post birth in rats. Recent studies, however, showed that SCs present in the transition region at the rete testis exhibit a more immature phenotype and have prolonged mitotic activity, which led to the hypothesis that SCs in this region will retain the capacity to respond to PTU treatment over a longer period of time. In the present study, male Wistar rats were treated with PTU from days 21 to 40 and were evaluated at 40 and 160 days of age. Similar to neonatal rat SCs, it was demonstrated that prepubertal SCs in the transition region have a high mitotic activity and are highly sensitive to TH levels. This delayed, transient hypothyroidism resulted in significantly increased testis weight, SCs number and daily sperm production. The results demonstrate for the first time that Sertoli cells showing plasticity in the transition region can be stimulated to increase proliferation and contribute to a late stage surge in testis weight and sperm output.

The effects of apelin on myometrium contractions in pregnant rats

Apelin, which is a new hormone, is secreted especially in the brain by hypothalamus as well as by many other organs like the stomach, fat tissue, and the heart. For apelin, whose possible effects on many bodily functions like the endocrine system, cardiovascular system and metabolic activities are still under investigation, the reproductive system is also an important target area. The purpose of the present study was to investigate the effects of plasma apelin levels in rats that were in diestrus, pregnancy and lactation periods, and to examine its possible effects on myometrium contractions of pregnant rats and non-pregnant rats that were in diestrus period. The plasma apelin concentrations in female adult Wistar rats were determined with the ELISA method in the diestrus period, and on the 12th, 18th, and 21st days of the pregnancy, and on the 2nd and 10th days of lactation (n=7 for each group). In addition, the effect of apelin at 0.01, 0.1, 1 and 10 μM doses on isometric contractions in the rat uterus on the 21st day of pregnancy and in diestrus period was tested by using isolated organ bath. This protocol was repeated under conditions that were pre-treated with protein kinase C inhibitor in calcium-free medium, and the possible effect of apelin on contractions and the mechanisms that might mediate this effect were investigated. When plasma apelin levels were compared with the rats in diestrus period, the apelin concentrations in the 21-day pregnancy group were high at a significant level (p&lt;0.05); and low at a significant level in the 2-day lactation group (p&lt;0.05). In myometrium contraction trials, it was observed that apelin induced the contractions. Apelin increased the frequency of the myometrium contractions at a significant level when applied at 1 μM and 10 μM concentrations (p&lt;0.05 and p&lt;0.001). Only after the apelin application at 10 μM concentration did the amplitude of the contractions increase at a significant level (p&lt;0.01). In the myometrium of the rats that were on the 21st day of pregnancy, the frequency of the contractions was statistically significant at 0.1 μM, 1 μM and 10 μM doses (p&lt;0.01). In addition, the amplitude of the contractions increased at a statistically significant level at 1 μM and 10 μM dose application (p&lt;0.05 and p&lt;0.01, respectively). The apelin application induced the contractions in calcium-free medium. When apelin was applied after the pre-application with protein kinase C inhibitor, no contractions were observed. The present study showed that apelin levels were increased at the end of pregnancy in rats, and the hormone induced the uterus contractions. This effect does not occur with protein kinase C inhibitor and in calcium-free medium, which shows that protein kinase C pathway might play a role in these mechanism. These findings show that apelin might be an endogenous peptide that plays a role on uterine contractions at birth in rats.

Apoptosis pattern and alterations of expression of apoptosis-related factors of supporting cells in Kölliker's organ in vivo in early stage after birth in rats.

Kolliker’s organ is a temporary but indispensable structure in the development of the cochlea. Supporting cells (SCs) within it release adenosine 5’-triphosphate (ATP), which may play a crucial role in cochlear development before the onset of hearing. To reveal the apoptosis of Kolliker’s organ in new-born rats, we studied the morphological changes and expression of apoptosis-related factors during early postnatal development. We found SCs in Kolliker’s organ decreased in number and changed in appearance along the cochlea apex-to-base gradient, and the expression of caspase-3, caspase-8, caspase- 9 and bcl-2 in Kolliker’s organ of the cochlea fluctuated along the course of postnatal development, with an expression peak at postnatal day 3. This study demonstrates a time-dependent degeneration of Kolliker’s organ during postnatal cochlea development, which might be triggered by endogenous factors.

Neonatal Desensitization Supports Long-Term Survival and Functional Integration of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Rat Joint Cartilage Without Immunosuppression

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models.

Abstract 171: Plasma Hypoosmolarity After Birth Promotes Closure of the Ductus Arteriosus

Background: Changes in environmental conditions after birth, such as increased oxygen tension, are known to regulate the closure of the ductus arteriosus (DA). We previously found that plasma osmolarity was significantly decreased early after birth in rats, and that Transient Receptor Potential Melastatin (TRPM) 3, a hypoosmolarity sensor, was highly expressed in the rat DA compared to the aorta. We assessed the hypothesis that plasma hypoosmolarity regulated tension of the DA via TRPM3. Methods: Fura-2 assay was used to evaluate the change in intracellular calcium [Ca 2+ ] i in vascular smooth muscle cells (SMCs). Tension of vascular rings was measured by a wire myograph system. Rapid whole-body freezing method was used to examine vasoconstriction in rat in vivo . Plasma osmolarity of human preterm infants was analyzed by freezing point depression osmometry. Results: We found that [Ca 2+ ] i was increased by hypoosmolarity (270mOsm/kg) in SMCs isolated from the rat DA (DASMCs) compared to aortic SMCs (31.3% vs. 8.5% of basal 340/390 ratio P &lt;0.01, n=6). When DASMCs were treated with siTRPM3, hypoosmolarity-induced [Ca 2+ ] i elevation was attenuated by 48.7% ( P &lt;0.01, n=6). Hypoosmolarity (270 and 250mOsm/kg) increased the tension of rat DA (17.4% and 29.8% of 120mM of KCl, respectively, P &lt;0.01, n=8). Moreover, a TRPM3 activator pregnenolone sulfate (200μ M) induced contraction of the rat DA (39.4% of KCl P &lt;0.001, n=6). Conversely, when plasma osmolarity was kept higher to 322mOsm/kg by intra-peritoneal injection of 5.4% hypersaline, the DA closure was partially inhibited by 13% compared to 0.9% saline injection in vivo ( P &lt;0.05, n=11). Plasma osmolarity was decreased 2 h after birth in late preterm infants (28-35weeks’ gestation) (3.2±0.8 % reduction of cord blood, P &lt;0.001, n=35), but not in early preterm infants (24-27weeks’ gestation) (1.1±1.3 % reduction of cord blood, NS , n=17). Furthermore, plasma osmolarity of patent DA (PDA) patients recovered to the adult level by the 2nd day of life while it still remained low in non PDA patients. Conclusions: These results suggest that plasma hypoosmolarity after birth promotes DA closure via TRPM3 and that keeping the plasma osmolarity within proper levels during early neonatal period would be important for DA closure.

Cardiac Injury Protection from Mouse Bone Marrow Stromal Cells with In Utero Transplantation Followed by Secondary Postnatal Boost

Limited donor-cell engraftment to the injured tissue restricts therapeutic efficacy of stem cell transplantation. Herein, we proposed an alternative strategy by using in utero transplantation (IUT) to create mixed-chimerism environment in recipients and to facilitate donor-cell engraftment followed by postnatal secondary boost with the same cells. Mouse bone marrow stromal cells (BMSCs) were used as the xenogenic donor cells and given into rat fetus as an early exposure of IUT treatment. The engraftment potential was analyzed for the presence of BMSCs by flow cytometry or PCR in recipient tissues. The function of a second boost of mouse BMSCs, in terms of cardioprotection, was tested by given 1×10⁶ cells to rat IUT hearts with ischemia/reperfusion (IR) injury that was induced by a 45 min of left coronary ligation and released for 72 h. Mouse BMSCs demonstrated an immunosuppressive effect when mixed with mouse or rat lymphocytes. IUT treatment only caused few BMSCs engrafted to fetal (embryonic day 20) and adult (4 weeks after birth) rat organs including heart, but engraftment was increased in hearts of the IUT rats after second boost. This was coincided with attenuation of cardiac injury caused by IR. Interestingly, an up-regulation of CXC chemokine receptor type 4 (CXCR4) was seen when BMSCs were exposed to hypoxia. This indicates that enhanced engraftment of mouse BMSCs to post-ischemic rat hearts possibly is dependent on CXCR4. Moreover, results of flow cytometry demonstrated that the presence of CD34⁺ cells in rat IUT hearts with IR injury was increased. These observations suggest that enhanced engraftment of donor BMSCs to rat IR hearts by CXCR4 may recruit endogenous CD34⁺ cells of recipients which in turn protects heart against IR. This also supports the notion of fetal preconditioning with BMSC enhances the efficiency of progenitor cell-mediated organ protection after a postnatal second boost in xeno-transplantation.

Obestatin and insulin in pancreas of newborn diabetic rats treated with exogenous ghrelin

The aim of the study was to evaluate the effect of ghrelin treatment on obestatin, insulin gene expression and biochemical parameters in the pancreas of newborn-streptozocin (STZ) diabetic rats. Rats were divided into 4 groups. Group I: control rats treated with physiological saline; group II: control rats treated with 100μg/kg/day ghrelin; group III: two days after birth rats that received 100mg/kg STZ injected as a single dose to induce neonatal diabetes; group IV: neonatal-STZ-diabetic rats treated with ghrelin for four weeks. Sections of the pancreas were examined with immunohistochemistry for the expression of obestatin and insulin and in situ hybridization for the expression of insulin mRNA. The blood glucose levels were measured. Tissue homogenates were used for protein, glutathione, lipid peroxidation and non-enzymatic glycosylation levels and antioxidant enzyme analysis. There was a significant difference in blood glucose levels in newborn-STZ-diabetic rats compared to ghrelin treated diabetic rats at weeks 1, 2 and 4. In group IV, pancreatic non-enzymatic glycosylation and lipid peroxidation levels were decreased, however, glutathione levels and enzymatic activities were increased. Insulin peptide and mRNA (+) signals in islets of Langerhans and obestatin immunopositive cell numbers showed an increase in group IV compared to group III. These results suggest that administration of ghrelin to newborn rats may prevent effects of diabetes.

Expression of Toll-like receptor-2 in premature rats with necrotizing enterocolitis and regulatory effects of glutamine

Objective To study the expression of Toll-like receptor-2 (TLR-2) and caspase-3 in the intestine of premature rats with necrotizing enterocolitis (NEC),and to explore the protective effects and possible regulatory mechanism of glutamine (Gln) in the NEC.Methods Sixty premature rats (gestational age 21 d) were divided into three groups (n = 20 each) according to the random number table: control group,model group and Gln intervention group.Rats in model group were given formula feeding,hypoxia and cold stress.Rats in Gln intervention group were given Gln 0.3 g/kg to the formula feeding,hypoxia and cold stress.All the premature rats were sacrificed and the intestine tissues were obtained on the third day after birth.The histological changes of ileal tissues were scored after HE staining.The expression of TLR-2 and caspase-3 in jejunum,ileum and colon were detected by inmunohistochemistry,and the expression of TLR-2 mRNA in jejunum,ileum and colon were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction.Results Pathology score of ileum in model group,Gln intervention group and control group were 3.10 ±0.99,2.40 ± 0.69 and 0.30 ±0.48,respectively.The expressions of TLR-2 protein in ileum were 2.53±0.94,2.15±0.82 and 1.57 ± 0.62 in the three groups respectively,and the expression of caspase-3 protein were 2.83 ± 0.45,2.70 ± 0.04 and 0.91 ± 0.29.The content of TLR2 mRNA in model group was 1.46 times higher than that of Gln intervention group and was 2.10 times higher than that of control group.Compared with the control group,the pathology score,expression of TLR-2 and caspase-3 protein,and TLR-2 mRNA in model group were significantly higher,P＜0.01.However,compared with the model group,those changes were improved in Gln intervention group,P＜0.05.Expression of TLR-2 mRNA positively correlated to the expression of caspase-3 protein (r=0.71,P＜0.01) and pathology score (r = 0.69,P＜ 0.01).Expression of caspase-3 protein positively correlated to the intestine injury pathology score (r=0.81,P＜0.01).Conclusions TLR-2 may be involved in the pathogenesis of NEC.Gln might reduce the expression of TLR-2 in the intestine,and decrease the apoptosis of intestinal epithelial cells to protect the intestine of preterm birth rats. Key words: Enterocolitis,necrotizing; Glutamine; Toll-like receptor 2; Apoptosis; Premature birth; Rats

Erythropoietin protects against apoptosis and increases expression of non‐neuronal cell markers in the hypoxia‐injured developing brain

Erythropoietin (EPO) is a cytokine hormone with cytoprotective effects in many tissues including the brain. Although the benefits of administration of recombinant human EPO (rhEPO) for neonatal hypoxic brain injury have been demonstrated in neuronal tissue, the effect on non-neuronal cell populations is unclear. We tested the hypothesis that rhEPO would not only protect neuronal cells but also glial cells at a stage of brain development where their maturation was particularly sensitive, and also protect the vasculature. This was evaluated in a rat model of hypoxic injury. 1000 IU/kg rhEPO was delivered intraperitoneally at the start of 4 h hypoxia or normoxia. Treatment groups of neonatal rats (day of birth, at least N = 10 per group) were as follows: normoxia; normoxia plus rhEPO; hypoxia (8% FiO(2) delivered in temperature-controlled chambers); and hypoxia plus rhEPO. Day of birth in rats is equivalent to human gestation of 28-32 weeks. The effects of rhEPO administration, especially to non-neuronal cell populations, and the associated molecular pathways, were investigated. Apoptosis was increased with hypoxia and this was significantly reduced with rhEPO (p < 0.05). The neuronal marker, microtubule-associated protein-2, increased in expression (p < 0.05) when apoptosis was significantly reduced by rhEPO. In addition, compared with hypoxia alone, rhEPO-treated hypoxia had the following significant protein expression increases (p < 0.05): the intermediate filament structural protein nestin; myelin basic protein (oligodendrocytes); and glial fibrillary acidic protein (astrocytes). In conclusion, rhEPO protects the developing brain via anti-apoptotic mechanisms and promotes the health of non-neuronal as well as neuronal cell populations at a time when loss of these cells would have long-lasting effects on brain function.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.