Respiratory sinus arrhythmia in spontaneously breathing, unanesthetized newborn and adult Wistar rats.

We examined respiratory sinus arrhythmia (RSA) and possible interaction with respiratory frequency (fR) and heart rate (HR) in spontaneously breathing, unanesthetized newborn Wistar rats (2- to 5-day-old; n = 54) and the adult rats (8-week-old; n = 34). Instantaneous heart rate (inst-HR) was calculated as the reciprocal of the inter-beat-interval. For each breath, RSA was determined as the difference between the maximum and minimum inst-HR value. The absolute RSA or RSA% (RSA per HR) were calculated as the average RSA of 10 consecutive breaths. RSA (or RSA%) in the newborn rats was significantly lower than that in the adult rats. Correlation coefficient between RSA (or RSA%) and 1/fR or HR/fR, but not HR, was significant in newborn rats, whereas only that between RSA (or RSA%) and HR was significant in adult rats. The power spectrum density of heartbeat fluctuation was detectable in both age groups. The present findings suggest that RSA exists and could be influenced by fR, rather than HR, in newborn rats.

Involvement of cannabinoid receptors in depression of the putative nociceptive response in spinal cord preparations isolated from neonatal rats.

A metabolite of acetaminophen, AM404, which is an anandamide transporter inhibitor, induces analgesia mainly via activation of transient receptor potential channel 1 in the spinal cord, although the role of cannabinoid receptors remains to be studied. The ventral root reflex response induced by stimulation of the dorsal root in in vitro preparations of rat spinal cord is useful to assess the effect of analgesics. We analyzed the effects of AM404 and cannabinoid receptor antagonist AM251 on reflex responses in lumbar spinal cord preparations from newborn rats and found that the amplitude of the slow ventral root potential after administration of 10 µM AM404 was not significantly changed, whereas 10 µM AM251 significantly increased the amplitude. Administration of the cannabinoid receptor 1 agonist WIN55,212-2 (10 µM) did not significantly affect the reflex response. We suggest that endogenous cannabinoids in the spinal cord are involved in the antinociceptive mechanism through suppressive effects.

Limiting Monoamines Degradation Increases L-DOPA Pro-Locomotor Action in Newborn Rats.

L-DOPA, the precursor of catecholamines, exerts a pro-locomotor action in several vertebrate species, including newborn rats. Here, we tested the hypothesis that decreasing the degradation of monoamines can promote the pro-locomotor action of a low, subthreshold dose of L-DOPA in five-day-old rats. The activity of the degrading pathways involving monoamine oxidases or catechol-O-methyltransferase was impaired by injecting nialamide or tolcapone, respectively. At this early post-natal stage, the capacity of the drugs to trigger locomotion was investigated by monitoring the air-stepping activity expressed by the animals suspended in a harness above the ground. We show that nialamide (100 mg/kg) or tolcapone (100 mg/kg), without effect on their own promotes maximal expression of air-stepping sequences in the presence of a sub-effective dose of L-DOPA (25 mg/kg). Tissue measurements of monoamines (dopamine, noradrenaline, serotonin and some of their metabolites) in the cervical and lumbar spinal cord confirmed the regional efficacy of each inhibitor toward their respective enzyme. Our experiments support the idea that the raise of monoamines boost L-DOPA's locomotor action. Considering that both inhibitors differently altered the spinal monoamines levels in response to L-DOPA, our data also suggest that maximal locomotor response can be reached with different monoamines environment.

Effects of aconitine on the respiratory activity of brainstem-spinal cord preparations isolated from newborn rats.

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5 µM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 µM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 µM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 µM riluzole or 100 µM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 µM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.

Cell Responses of the Ventrolateral Medulla to PAR1 Activation and Changes in Respiratory Rhythm in Newborn Rat En Bloc Brainstem-Spinal Cord Preparations.

Proteinase-activated receptor-1 (PAR1) is expressed in astrocytes of various brain regions, and its activation is involved in the modulation of neuronal activity. Here, we report effects of PAR1 selective agonist TFLLR on respiratory rhythm generation in brainstem-spinal cord preparations. Preparations were isolated from newborn rats (P0-P4) under deep isoflurane anesthesia and were transversely cut at the rostral medulla. Preparations were superfused with artificial cerebrospinal fluid (25-26 °C), and inspiratory C4 ventral root activity was monitored. The responses to TFLLR of cells close to the cut surface were detected by calcium imaging or membrane potential recordings. Application of 10 µM TFLLR (4 min) induced a rapid and transient increase of calcium signal in cells of the ventrolateral respiratory regions of the medulla. More than 88% of responding cells (223/254 cells from 13 preparations) were also activated by low (0.2 mM) K+ solution, suggesting that they were astrocytes. Immunohistochemical examination demonstrated that PAR1 was expressed on many astrocytes. Respiratory-related neurons in the medulla were transiently hyperpolarized (-1.8 mV) during 10 µM TFLLR application, followed by weak membrane depolarization after washout. C4 burst rate decreased transiently in response to application of TFLLR, followed by a slight increase. The inhibitory effect was partially blocked by 50 µM theophylline. In conclusion, activation of astrocytes via PAR1 resulted in a decrease of inspiratory C4 burst rate in association with transient hyperpolarization of respiratory-related neurons. After washout, slow and weak excitatory responses appeared. Adenosine may be partially involved in the inhibitory effect of PAR1 activation.

Dexmedetomidine Protects Cerebellar Neurons against Hyperoxia-Induced Oxidative Stress and Apoptosis in the Juvenile Rat.

The risk of oxidative stress is unavoidable in preterm infants and increases the risk of neonatal morbidities. Premature infants often require sedation and analgesia, and the commonly used opioids and benzodiazepines are associated with adverse effects. Impairment of cerebellar functions during cognitive development could be a crucial factor in neurodevelopmental disorders of prematurity. Recent studies have focused on dexmedetomidine (DEX), which has been associated with potential neuroprotective properties and is used as an off-label application in neonatal units. Wistar rats (P6) were exposed to 80% hyperoxia for 24 h and received as pretreatment a single dose of DEX (5µg/kg, i.p.). Analyses in the immature rat cerebellum immediately after hyperoxia (P7) and after recovery to room air (P9, P11, and P14) included examinations for cell death and inflammatory and oxidative responses. Acute exposure to high oxygen concentrations caused a significant oxidative stress response, with a return to normal levels by P14. A marked reduction of hyperoxia-mediated damage was demonstrated after DEX pretreatment. DEX produced a much earlier recovery than in controls, confirming a neuroprotective effect of DEX on alterations elicited by oxygen stress on the developing cerebellum.

Protective Effect of Dexmedetomidine against Hyperoxia-Damaged Cerebellar Neurodevelopment in the Juvenile Rat.

Impaired cerebellar development of premature infants and the associated impairment of cerebellar functions in cognitive development could be crucial factors for neurodevelopmental disorders. Anesthetic- and hyperoxia-induced neurotoxicity of the immature brain can lead to learning and behavioral disorders. Dexmedetomidine (DEX), which is associated with neuroprotective properties, is increasingly being studied for off-label use in the NICU. For this purpose, six-day-old Wistar rats (P6) were exposed to hyperoxia (80% O2) or normoxia (21% O2) for 24 h after DEX (5 µg/kg, i.p.) or vehicle (0.9% NaCl) application. An initial detection in the immature rat cerebellum was performed after the termination of hyperoxia at P7 and then after recovery in room air at P9, P11, and P14. Hyperoxia reduced the proportion of Calb1+-Purkinje cells and affected the dendrite length at P7 and/or P9/P11. Proliferating Pax6+-granule progenitors remained reduced after hyperoxia and until P14. The expression of neurotrophins and neuronal transcription factors/markers of proliferation, migration, and survival were also reduced by oxidative stress in different manners. DEX demonstrated protective effects on hyperoxia-injured Purkinje cells, and DEX without hyperoxia modulated neuronal transcription in the short term without any effects at the cellular level. DEX protects hyperoxia-damaged Purkinje cells and appears to differentially affect cerebellar granular cell neurogenesis following oxidative stress.

Protective Effects of Early Caffeine Administration in Hyperoxia-Induced Neurotoxicity in the Juvenile Rat.

High-risk preterm infants are affected by a higher incidence of cognitive developmental deficits due to the unavoidable risk factor of oxygen toxicity. Caffeine is known to have a protective effect in preventing bronchopulmonary dysplasia associated with improved neurologic outcomes, although very early initiation of therapy is controversial. In this study, we used newborn rats in an oxygen injury model to test the hypothesis that near-birth caffeine administration modulates neuronal maturation and differentiation in the hippocampus of the developing brain. For this purpose, newborn Wistar rats were exposed to 21% or 80% oxygen on the day of birth for 3 or 5 days and treated with vehicle or caffeine (10 mg/kg/48 h). Postnatal exposure to 80% oxygen resulted in a drastic reduction of associated neuronal mediators for radial glia, mitotic/postmitotic neurons, and impaired cell-cycle regulation, predominantly persistent even after recovery to room air until postnatal day 15. Systemic caffeine administration significantly counteracted the effects of oxygen insult on neuronal maturation in the hippocampus. Interestingly, under normoxia, caffeine inhibited the transcription of neuronal mediators of maturing and mature neurons. The early administration of caffeine modulated hyperoxia-induced decreased neurogenesis in the hippocampus and showed neuroprotective properties in the neonatal rat oxygen toxicity model.

Preventive effects of antenatal CDP-choline in a rat model of neonatal hyperoxia-induced lung injury.

Antenatal steroid administration to pregnant women at risk of prematurity provides pulmonary maturation in infants, while it has limited effects on incidence of bronchopulmonary dysplasia (BPD), the clinical expression of hyperoxia-induced lung injury (HILI). Cytidine-5'-diphosphate choline (CDP-choline) was shown to alleviate HILI when administered to newborn rats. Therefore, we investigated effects of maternal administration of CDP-choline, alone or in combination with betamethasone, on lung maturation in neonatal rats subjected to HILI immediately after birth. Pregnant rats were randomly assigned to one of the four treatments: saline (1 mL/kg), CDP-choline (300 mg/kg), betamethasone (0.4 mg/kg), or CDP-choline plus betamethasone (combination therapy). From postnatal day 1 to 11, pups born to mothers in the same treatment group were pooled and randomly assigned to either normoxia or hyperoxia group. Biochemical an d histopathological effects of CDP-choline on neonatal lung tissue were evaluated. Antenatal CDP-choline treatment increased levels of phosphatidylcholine and total lung phospholipids, decreased apoptosis, and improved alveolarization. The outcomes were further improved with combination therapy compared to the administration of CDP-choline or betamethasone alone. These results demonstrate that antenatal CDP-choline treatment provides benefit in experimental HILI either alone or more intensively when administered along with a steroid, suggesting a possible utility for CDP-choline against BPD.

Effect of cisplatin on respiratory activity in neonatal rats.

One side effect of cisplatin, a cytotoxic platinum anticancer drug, is peripheral neuropathy; however, its central nervous system effects remain unclear. We monitored respiratory nerve activity from the C4 ventral root in brainstem and spinal cord preparations from neonatal rats (P0-3) to investigate its central effects. Bath application of 10-100 µM cisplatin for 15-20 min dose-dependently decreased the respiratory rate and increased the amplitude of C4 inspiratory activity. These effects were not reversed after washout. In separate perfusion experiments, cisplatin application to the medulla decreased the respiratory rate, and application to the spinal cord increased the C4 burst amplitude without changing the burst rate. Application of other platinum drugs, carboplatin or oxaliplatin, induced no change of respiratory activity. A membrane potential analysis of respiratory-related neurons in the rostral medulla showed that firing frequencies of action potentials in the burst phase tended to decrease during cisplatin application. In contrast, in inspiratory spinal motor neurons, cisplatin application increased the peak firing frequency of action potentials during the inspiratory burst phase. The increased burst amplitude and decreased respiratory frequency were partially antagonized by riluzole and picrotoxin, respectively. Taken together, cisplatin inhibited respiratory rhythm via medullary inhibitory system activation and enhanced inspiratory motor nerve activity by changing the firing property of motor neurons.

The expression of miR-876-3p based on a rat model of bronchopulmonary dysplasia induced by hyperoxia / 中国小儿急救医学

Objective:To establish a neonatal rat bronchopulmonary dysplasia(BPD) model induced by hyperoxia, to detect the expression of miR-876-3p in the lung tissue, and to analyze the role of miR-876-3p in the occurrence and development of BPD, so as to provide a theoretical basis for the pathogenesis, prevention and treatment of BPD.Methods:Eighty newborn SD rats were randomly divided into hyperoxia group(FiO 2 60%) and air group(FiO 2 21%). Lung tissue samples were taken on the 1st, 7th, 14th and 21st day after birth, the pathological changes of lung tissue were observed.Quantitative real-time PCR technique was used to detect the expression level of miR-876-3p. Results:Within 21 days after birth, with the prolongation of hyperoxia exposure time, the general growth of rats in hyperoxia group were lower than those in air group[14 d: (35.46±1.62) g vs.(37.08±1.25) g; 21 d: (51.92±1.83) g vs.(58.87±2.43) g]( P<0.05). On the 14th and 21st day after birth, the radial alveolar counts in lung tissue of rats in hyperoxia group were significantly reduced compared with those in air group( P<0.05). On the 7th, 14th and 21st day after birth, the alveolar septal thickness of rats in air group were lower than those in hyperoxia group( P<0.05). The expression level of miR-876-3p in hyperoxia group decreased gradually and was significantly lower on the 7th, 14th and 21st day compared with air group at the same time points[7 d: (14.97±1.13) vs.(16.64±0.89); 14 d: (11.92±0.71) vs.(16.85±0.79); 21 d: (11.39±0.79) vs.(17.52±1.17)], and the differences were all statistically significant( P all<0.01). Conclusion:In this study, a new BPD model of neonatal rats can be induced by hyperoxia and the expression level of miR-876-3p in this model is decreased.The differential expression level of miR-876-3p may play a role in the occurrence and development of BPD.

Age-dependent effect of insulin in the regulation of intracellular calcium in ventricular cardiomyocytes.

In this study, we wanted to verify whether the effect of insulin on calcium homeostasis depends on the heart's development stage. Using a quantitative 3D confocal microscopy, we tested the effect of a high insulin concentration (100 µU) in freshly cultured ventricular cardiomyocytes from newborn and adult rats. Our results showed that the cytosolic basal level of calcium was higher in newborn cardiomyocytes with no change in the nuclear basal calcium level compared with the adult cardiomyocytes; in addition, insulin induced a slow increase of cytosolic and nuclear calcium in newborn ventricular cardiomyocytes, followed by two phases. However, the first phase of slow cytosolic and nuclear calcium increase was absent in adult rat ventricular cardiomyocytes. Furthermore, the time to the onset of increase of cytosolic and nuclear calcium was longer in newborn cardiomyocytes compared with adults. Moreover, the time to peak of the calcium transient was shorter in newborns than in adult cardiomyocytes. These results demonstrate that insulin differently regulates calcium homeostasis in newborns than in adult cardiomyocytes. Thus, newborn rat cardiomyocytes, commonly used in research as a model for adult cardiomyocytes, should be used with caution when dealing with insulin in normal and disease conditions.

Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat.

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

The expression and effect of DNA damage repair protein NBS1 in neonatal rats with bronchopulmonary dysplasia / 国际儿科学杂志

Objective:To investigate the dynamic expression of DNA damage repair protein Nijmegen breakage syndrome protein 1(NBS1) in the neonatal rats with bronchopulmonary dysplasia(BPD), and its influence on the development and progression of BPD.Methods:Newborn rats were randomly divided into the BPD model group( n=50) and the control group( n=50) within 12 h after birth.The inhaled oxygen concentration was 80%-85% in the model group, and the control group inhaled air.In the two groups, lung tissue samples were collected on days 1, 3, 7, 10 and 14, and isolated, purified and cultured alveolar epithelial type Ⅱ cells(AEC Ⅱ). We observed pulmonary morphological changes under light microscope and evaluated alveolar development degree by radiate alveolar counts(RAC). Immunohistochemistry and cell immunofluorescence were used to observe the localization and expression of NBS1.Western blot and real-time quantitative PCR were used to detect the expression level of NBS1. Results:Compared with the control group, the RAC value in the model group was decreased significantly from 7 d after birth(control group 7.58±1.24, model group 5.42±1.24, P<0.01). Immunohistochemistry showed that NBS1 protein was mainly located in the nucleus of alveolar epithelial cells.In the model group, NBS1 was mainly expressed in the nucleus on the 1st day.With the prolonged exposure time, the number of cytoplasmic staining cells increased and the expression in the nucleus decreased.Cell immunofluorescence farther showed that NBS1 protein was mainly located in the nucleus in AEC Ⅱ.Compared with the control group, cytoplasmic staining in model group was enhanced from 3 d, while nuclear staining was gradually weakened, and was mainly located in the cytoplasm at 14 d. Western blot results showed that the expression of NBS1 protein in the model group peaked at 1 d compared to the control group(control group 0.72±0.29, model group 1.28±0.22, P<0.01), and then gradually decreased, with lower expression at 14 d compared to the control group(control group 0.73±0.19, model group 0.49±0.11, P<0.05). Similarly, the mRNA expression level of NBS1 in the model group peaked at 1 d compared to the control group(control group 1.00±0.00, model group 1.18±0.06, P<0.01), and then gradually decreased, with lower expression at 14 d than that in the control group(control group 1.07±0.13, model group 0.76±0.11, P<0.05). Conclusion:In the neonatal rats with BPD, the down-regulation expression and nuclear enrichment disorder of NBS1 may affect the DNA damage response and be one of the mechanisms mediating the onset of oxidative stress damage in BPD.

Effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury in newborn rats.

To explore the effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury (HIBI) in newborn rats. Seventy-two healthy SPF grade SD newborn rats were randomly and equally divided into Normal group (healthy rats) and Sham group (rats underwent sham operation), Model group (HIBI model rats), p38 MAPK Inhibitor group (HIBI model rats treated with p38 MAPK inhibitor) and p38 MAPK Activator group (HIBI model rats treated with p38 MAPK activator). On postnatal day 28, Morris water maze, tail suspension test and inclined plane test were conducted on rats in each group. Twenty-four hours after modeling, the expression of p-p38 MAPK protein and apoptosis related genes in rat hippocampal tissues was detected by TUNEL staining, qRT-PCR and Western blot. Compared with Normal group, escape latency and inclined plane test time were prolonged, the number of passing through the platform and tail suspension time were reduced (all P < 0.05); Bax and Caspase-3 mRNA and protein expression levels and p-p38 MAPK protein level were increased, Bcl-2 mRNA level was decreased, and neuronal apoptosis proportion was increased in Model group (all P < 0.05). Compared with Model group, the above indicators showed reversed and enhanced trends in p38 MAPK Inhibitor and p38 MAPK Activator groups, respectively (all P < 0.05). Inhibition of p38 MAPK signaling pathway can effectively improve the learning and memory ability and motor function of newborn rats with HIBI, and reduce neuronal apoptosis in the hippocampal tissues, thereby promoting neuronal recovery.

Antenatal Endotoxin Impairs Lung Mechanics and Increases Sensitivity to Ventilator-Induced Lung Injury in Newborn Rat Pups.

Perinatal inflammation due to chorioamnionitis and ventilator-induced lung injury (VILI) at birth is independent risk factors for the development of bronchopulmonary dysplasia (BPD). We have previously shown that antenatal endotoxin (ETX) causes abnormal lung structure and function in 2-week-old rats, but whether ETX impairs lung mechanics at birth and increases risk for VILI is unknown. Fetal rats were exposed to 10 µg endotoxin or saline via intra-amniotic injection. At birth (D0) or 7 days (D7), rats received 90 min of lung protective ventilation [PROTECT group; tidal volume (Vt) = 6 ml/kg with positive end expiratory pressure (PEEP) = 2 cmH2O]; P20 ventilation [plateau pressure (Pplat) = 20 cmH2O, PEEP = 0]; or P24 ventilation (Pplat = 24 cmH2O, PEEP = 0, only applied to D7). Prior to prolonged ventilation at D0, endotoxin-exposed rats had decreased compliance and inspiratory capacity (IC) compared to controls. At D7, endotoxin was associated with reduced compliance. High-pressure ventilation (P20 and P24) tended to increase IC and compliance in all saline-treated groups. Ventilation at D0 with P20 increased IC and compliance when applied to saline-treated but not endotoxin-exposed pups. At D7, P24 ventilation of endotoxin-exposed pups increased elastance, bronchoalveolar lavage protein content, and IL-1b and TEN-C mRNA expression in comparison to the saline group. In summary, antenatal endotoxin exposure alters lung mechanics at birth and 1 week of life and increases susceptibility to VILI as observed in lung mechanics, alveolocapillary barrier injury, and inflammatory mRNA expression. We speculate that antenatal inflammation primes the lung for a more marked VILI response, suggesting an adverse synergistic effect of antenatal and postnatal exposures.

Neuroprotective effect of L-arginine in a neonatal rat model of hypoxic-ischemia.

Aim: The aim of the present study is to investigate the neuroprotective effects of l-Arginine (l-arg) in the seven-day-old rat hypoxia-ischemia model. Materials and methods: L-Arginine (n = 10) or saline (n = 8) was administered intraperitoneally to seven-day-old rats before hypoxia-ischemia. In addition, 18 seven-day-old rats were given l-Arginine (n = 10) or saline (n = 8) after hypoxic-ischemic insult. Neuronal apoptosis was investigated by terminal dUDP-biotin nick end-labeling (TUNEL) following three days of recovery. The ratios of right side numerical density to the sum of right and left sides' numerical densities (right apoptosis index) were calculated for every brain region in rats receiving l-arginine and they were compared with the vehicle groups. Results: Right side apoptosis indexes of the hippocampus (mean ± SD; 35.0 ± 16.1) and striatum (41.9 ± 16.0) were significantly decreased in the l-Arginine post-treatment groups when compared to vehicles (61.0 ± 17.0 and 62.4 ± 27.0 respectively) (p < 0.05). There was no significant difference in the right apoptosis indexes of the cortex between l-Arginine post-treated group and the vehicle group. There were also no significant differences between the right side apoptosis indexes of the l-Arginine pretreatment groups and those of the vehicle group in any of the three regions (p > 0.05). Conclusions: It is concluded that neuronal apoptosis due to hypoxic-ischemic injury may likely to be reduced by post-treatment of l-Arginine in the neonatal rat model and l-Arginine provides a new possibility for neuroprotective strategies based on NO production.

The expression and effect of long non-coding RNA H19 in BPD neonatal rats / 国际儿科学杂志

Objective To investigate the expression of long non-coding RNA H19 (LncRNA H19)and its regulation of histone methyltransferase 2 (enhancer of zeste homolog 2,EZH2) in the lung tissue of neonatal rats with bronchopulmonary dysplasia (BPD),and to lay a foundation for elucidating the pathogenesis of BPD lung epithelium-interstitial transformation (EMT).Methods The BPD model of SD neonatal rats was induced by hyperoxia (inhalation oxygen concentration was 85％) (n =50),and oxygen inhalation concentration of the control group was 21％ (n =50).The two groups were collected at ld,3d,7d,14d and 21d after birth in lung tissue.Immunohistochemistry,Western blot,real-time quantitative PCR and other techniques were used to detect the intracellular localization,and the expression level of EZH2 protein and the mRNA expression level of H19 and EZH2.Results Immunohistochemistry showed that EZH2 protein was located in the nucleus and cytoplasm of alveolar epithelial cells,and the expression of EZH2 protein in the model group was significantly enhanced compared with the control group.Similarly,the results of Western blot demonstrated that the expression of EZH2 protein in the model group increased from ld (control group:0.196 ± 0.030,model group 0.650 ±0.149) to 21d (control group 0.934 ± 0.215,model group 1.785 ± 0.298) rather than the control group (P ＜ 0.05).Compared with the control group,the mRNA expression level of H19 in the model group increased from 7d (control group 2.591 ± 0.211,model group 3.558 ± 0.093,P ＜ 0.05) and the expression level of EZH2 mRNA started to increase from 3d (control group 1.246 ±0.015,model group 2.148 ± 0.215,P ＜0.05).Moreover,the differences between the two groups were obvious with the time of hyperoxia exposure.Conclusion In the development of BPD,the expression levels of H19 and EZH2 protein in lung tissue is up-regulated,and the peak of H19 expression precedes EZH2,which suggest that H19 might be involved in the pathogenesis of pulmonary dysplasia induced by EZH2-mediated EMT.

The expression and effect of TRF1 and TRF2 in neonatal rats with bronchopulmonary dysplasia / 国际儿科学杂志

Objective To investigate the dynamic expression of telomere repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) in the development and progression of bronchopulmonary dysplasia (BPD) in neonatal rats and to clarify its role in BPD alveolar dysplasia.Methods The neonatal rat BPD model (n =40) was induced by using neonatal SD rats with inhaled oxygen concentration of 85％.The control group was prepared by inhaled air (n =40).In the two groups,10 rats were randomly selected from 1 day,3 days,7 days,and 14 days after the experiment.The lung tissue samples were collected,HE staining was performed to observe the pathological changes,and the alveolar development degree was evaluated by radial alveolar counting (RAC).Immunohistochemistry was used to observe the localization and expression of TRF1 and TRF2.Western Blot and real-time quantitative PCR (RT-PCR) were used to detect the expression levels of TRF1 and TRF2 proteins and genes in lung tissue.Results Immunohistochemical staining showed that TRF1 was mainly localized in the nucleus of alveolar epithelial cells and bronchial epithelial cells.TRF2 protein was found in the nucleus and cytoplasm of alveolar epithelial cells and bronchial epithelial cells.The expression was significantly higher than that of the control group.Compared with the control group,the TRF1 and TRF2 proteins increased significantly from 1d (TRF1 in control group:0.163 ±0.022,in model group:0.251 ±0.022;TRF2 in control group:0.156 ±0.012,in model group:0.240 ±0.018) to 14d (TRF1 in control group:0.193 ± 0.024,in model group:0.230 ± 0.025;TRF2 in control group:0.225 ± 0.017,in model group:0.350 ±0.012) rather than the control group (P ＜ 0.05).The mRNA expression levels of TRF1 and TRF2 increased continuously from 1d to 7d of hyperoxia exposure (TRF1 in control group:0.946 ± 0.028,in model group:1.590 ± 0.228;TRF2 in control group:0.834 ± 0.083,in model group:1.783 ±0.262) and decreased at 14d (TRF1 in control group:2.217 ± 0.225,in model group:1.259 ± 0.217,P＜0.05;TRF2 in control group:2.143 ±0.250,in model group:0.997 ±0.199,P ＜0.05).Conclusion In the developmental stage of BPD,TRF1 and TRF2 act as negative regulators of telomere length,and protein levels are up-regulated,which suggest that they be involved in the pathological process of BPD alveolar dysplasia.

The combined effects of three-dimensional cell culture and natural tissue extract on neural differentiation of P19 embryonal carcinoma stem cells.

Tissue engineering, as a novel transplantation therapy, aims to create biomaterial scaffolds resembling the extracellular matrix in order to regenerate the damaged tissues. Adding bioactive factors to the scaffold would improve cell-tissue interactions. In this study, the effect of chitosan polyvinyl alcohol nanofibres containing carbon nanotube scaffold with or without active bioglass (BG+ /BG- ), in combination with neonatal rat brain extract on cell viability, proliferation, and neural differentiation of P19 embryonic carcinoma stem cells was investigated. To induce differentiation, the cells were cultured in α-MEM supplemented with neonatal rat brain extract on the scaffolds. The expression of undifferentiated stem cell markers as well as neuroepithelial and neural-specific markers was evaluated and confirmed by real-time Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence procedures. Finally, the three-dimensional (3D) cultured cells were implanted into the damaged neural tubes of chick embryos, and their fates were followed in ovo. Based on the histological and immunofluorescence observations, the transplanted cells were able to survive, migrate, and penetrate into the host embryonic tissues. Gene network analysis suggested the possible involvement of neurotransmitters as a downstream target of synaptophysin and tyrosine hydroxylase. Overall, the results of this study indicated that combining the effects of 3D cell culture and natural brain tissue extract can accelerate the differentiation of P19 embryonic carcinoma cells into neuronal phenotype cells.