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Objective:To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.Methods:Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined.Results:Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain. Conclusions:Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.
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Aims: Morphine treatment in early life is a practice widely used in paediatric intensive care units. However, the consequences of this treatment on behavioural responses throughout life have been poorly studied. It is well known that behavioural symptoms after morphine exposure are presumed to depend on certain changes in the dopaminergic system, and there is a strong evidence of the involvement of D2 receptor. In this way, our objective was to evaluate whether 5 μg morphine administration, once daily for 7 days in 8-day-old rats (P8), alters exploratory and anxiolitic-like behaviours over short- (P16) and medium-term (P30) periods in the open-field (OF) and elevated-plus maze (EPM) tests, and to verify the involvement of the D2 receptor in the behaviour changes. Place of Study: All experiments were performed at Animal Experimentation Unit of Hospital de Clínicas de Porto Alegre. The experimental protocol was approved by the Institutional Committee for Animal Care and Use (GPPG-HCPA protocol No: 08345). Methodology: Wistar rats with 8-day-old (P8) received 5 μg of morphine administration, once daily for 7 days. The exploratory and anxiolitic-like behavious were analyzed in P16 and P30 by OF and EPM tests. At the ages where we observed significant differences in behaviour responses in both tests, the control and morphine groups were subdivided into three groups, each one designed to evaluate the effect of 0.2 mg/kg, 0.5 mg/kg or vehicle of i.p. administration of a dopamine D2 antagonist receptor (Haloperidol). Results: At short-term (P16) morphine group showed an increase in grooming, as well increase in exploratory behaviours at P30 in the OF test. In addition, anxiolytic-like behaviours were observed in the morphine group, such as increase of percentage of open arms behaviours and non-protected head dipping at medium-term in the EPM test. At the ages at which differences in behaviours were observed, both groups (control and morphine) received D2 antagonist receptor (haloperidol) 30 min before each test. All behaviours changes seen in the morphine group at P30 were totally reversed by haloperidol administration. Conclusion: Our findings demonstrate that morphine treatment in neonate period promotes behavioural changes in OF and EPM at P16 and P30. However, only alterations observed at P30 depend, at least in part, of dopaminergic system, particularly of the D2 receptor.
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Objective: To study the protective effect of Danhong Injection (DI) on primary cultured neonate rat brain microvascular endothelial cells (rBMECs) injury. Methods: The primary cultured rBMEC model was established and the identification of rabbit anti rat VIII factor was carried out. The cells were divided into control, model, low-, mid-, and high-dose (25, 50, and 100 μL/mL) DI groups in hypoxic condition for 4 h after administration. The cell morphology was observed under microscope, the apoptosis rate and DNA content were determined by flow cytometry, and the lactate dehydrogenase (LDH) level in cultural supernatants and cell superoxide dismutase (SOD) activity were detected according to the kit methods. Results: DI (50 and 100 μL/mL) could alleviate the rBMEC damage induced by hypoxia remarkably, improve the cell morphology of rBMECs, decrease the apoptosis significantly, inhibit the blockage of rBMECs in G1/S phase and the leakage of LDH, and increase the SOD activity. Conclusion: DI plays a significant role in the protection on injured primary cultured rBMECs induced by hypoxia, and the mechanism may be related to the enhancement of cellular anti-oxidative capacity and the inhibition of apoptosis.
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Role of serotonin in olfactory recognition was tested by depleting the olfactory bulb serotonin during postnatal day (PND)1- 4 following administration of 5,7-dihydroxytryptamine. Significant difference in the olfactory recognition test was observed during PND5-7; control pups successfully recognized and oriented towards their mother; whereas treated pups failed to recognize their mother odour. Later on, during PND12-14, both group of pups responded equally in the recognition test. Levels of olfactory bulb serotonin were depleted (53.3%) in the treated pups on PND-8, which was restored on PND-14 with only 15% variation. Further analysis demonstrated that depletion of serotonin in olfactory bulb did not affect the normal suckling and weight gain, it only modulates olfactory recognition.
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Objective To investigate morphologic, structural and functional characteristics of cardiomyocytes from neonate rats and to set up a desirable technique for isolating and purifying the cardiomyocytes from neonate rats. Methods Using trypsin-digestion, mechanical separation, twice seedings and bromodeoxyuridine (BrdU)-treatment, the cardiomyocytes were isolated and purified from the hearts of neonate rats at 1-7 days after birth. Shapes and spontaneous pulsation of the cells were viewed. The cells were identified with cardiac isoform of tropnin T(cTnT) immunostaining. Ultrastructural features of the cells were examined with in situ transmission electron microscopy. Responses of the cells to adenine and isoprenaline were also examined. Results More than 95% cells isolated from the hearts of neonate rats are cardiomyocytes. The vital cells are more than 95%. Neonate rat cardiomyocytes include short columnar or rhabdoid cells and irregular cells. The most rhabdoid cells from the rats at 1-3 days after birth present the ultrastructural features of immature cardiomyocytes. The rhabdoid cells from the rats at 6-7 days after birth have some ultrastructural features of mature cardiomyocytes. Comparing with the cells at 1-3 days after birth, cTnT expression in the cells is slightly enhanced, the transverse striation was obvious. The irregular cells contain less bundles of myofilaments, the filaments are arranged irregularly. There are a few small cells which are in undifferentiated state. More than 80% cells show spontaneous pulsation at 72 hours after incubation. After treatment with adrenine and isoprenaline, the number of the cells with spontaneous pulsation increases and the intension of spontaneous pulsation is enhanced. The responses of the rhabdoid cells from the rats at 6-7 days after birth to adenine and isoprenaline are much stronger. Conclusion There are two kinds of neonate rat cardiomyocytes. They are different in ultrastructures, spontaneous pulsation and responses to adenine and isoprenaline. The cardiomyocytes from rats at 6-7 days after birth are suitable for experiments in vitro as mature cardiomyocyte. The method set up in this experiment is desirable for culture of neonate rat cardiomyocytes.
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Objective To establish a newborn animal model of renal injury caused by intrauterine asphyxia and explore the mechanism of renal injury in neonate after asphyxia.Methods After two-horn uterus and vessels supplying uterus and ovary were exposed in 21-day-pregnant Wistar rats,arterial clamp occluded one side of vessels.The occluding time were 10 and 30 minutes.Then arterial clamp was taken off,and reperfusion for 30 minutes,2,6,12 and 24 hours respectively.Reaching prescribed time uterus horn was opened rapidly and pups were removed.The pups sacrificed by decapitation.Kidneys were taken out and studied by HE staining and electron microscope.Results Kidney of fetal rats in 21 gestational age was developmental and mature degree of tubules dropped behind that of glomerule.Changes of proximal tubules were early and serious compared with distal tubules during ischemia and reperfusion stages.Conclusion Ischemia and reperfusion to graded pregnant rat can supply an ideal model to study injury of kidney and other organs(intraute)-rously.