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Objective To establish a simple and efficient method for isolation and culture of adult rat cardiac micro-vascular endothelial cells. Methods Wistar rats were anesthetized and thoracotomy was performed. Cardiac micro-vascular endothelial cells were isolated by heart perfusion and enzyme digestion. The morphology and growth curve of the third generation cells were examined by inverted microscopy. Its molecular markers factor Ⅷ-related antigen and CD31 were detected by immunocytochemistry and immunofluoresence. The angiogenic ability was examined by matrigel. Results The primary cultured cardiac microvascular endothelial cells in vitro were polygonal or fusiform with typical paving stone-like growth characteristics. At the same time,the cells were positive stained by factorⅧ-related antigen and CD31 and showed angiogenic ability. Conclusions Cardiac microvascular endothelial cells cul-tured by this method possess high purity. The method is easy to operate and can be used for laboratory and clinical research of cardiovascular diseases.
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Objective To measure and compare sevoflurane minimum alveolar concentration (MAC)in newborn and adult rats.Methods The rats were divided into newborn rat (6-8 days) group (25 cases)and adult rat (8-10 weeks)group (25 cases).All rats were settled in the self-made device for anesthesia with inhaled sevoflurane,and the tails of rats were exposed out of the device. Sevoflurane was given via an anesthesia machine.Up-and-down method and clamping tail stimulus were applied to measure the MAC values of sevoflurane in the two groups.1.5% of sevoflurane was set as the initial concentration,and± 0.2% as adjustable gradient.A positive or negative response was judged by clamping tail stimulus in a 20 minutes interval.The mean MAC value of all rats in each group was defined as the MAC value.Results The MAC value of newborn rats was (2.58 ± 0.1 1)%,and the MAC value of adult rats was (2.32 ±0.13)%.A significant difference was found between the two groups (P <0.01).Conclusion The MAC value of newborn rats was much higher than that of adult rats.Newborn rats need a higher concentration of sevoflurane at the same depth of anesthesia when compared with adult rats.
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Aim To screen the differentially expressed microRNAs ( miRNAs ) induced by hypoxia precondi-tioning ( HPC ) in adult rat cardiomyocytes, and pre-dict miRNAs-regulated target genes and their func-tions. Methods Cardiomyocytes were isolated from a-dult rat ventricular myocardium and cultured ( in vitro) . The cells were divided into 2 groups: control group ( CON ) and hypoxia preconditioning group ( HPC) . The cardiomyocytes in HPC group were sub-jected to 10 min hypoxia followed by 30 min reoxygen-ation, while the cells in CON group were cultured un-der normal condition. After that, total RNA was ex-tracted and then subjected to miRNA microarray to screen differentially expressed miRNAs. The microar-ray results were further validated by quantitative RT-PCR ( qRT-PCR ) . Bioinformatics analysis was per-formed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology ( GO) and sig-naling pathway ( Pathway) . Results HPC caused sig-nificant changes in miRNAs expression in cardiomyo-cytes as compared to CON group. A total of 12 miR-NAs were up-regulated and 14 miRNAs were down-reg-ulated ( P 500 were selected for further bioinformatics analysis. The expression of miR-133b-5p, miR-664-1-5p and miR-6216 detected by qRT-PCR exhibited the similar patterns of up or down regulation to those shown in mi-croarray results. Bioinformatics analysis revealed that miRNAs-regulated target genes were significantly en-riched in 27 GOs and 6 signal pathways. Conclusion The expression profile of miRNAs in rat cardiomyo-cytes is significantly affected by HPC. These differenti-ally expressed miRNAs might participate in HPC-in-duced cardioprotection by regulating their target genes in rat cardiomyocytes.
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Objective To compare two separation medium of isolation of adult rat cardiomyocytes , and to observe the characteristics of excitation-contraction coupling of cardiomyocytes .Methods The isolated adult rat heart was hanged on to the Langendorff apparatus for aortic counter-current perfusion and collagenase digestion using two different separation medium.The single cardiomyocytes were cultured and infected with adenovirus . The morphological features of cardiomyocytes were observed with microscope and fluorescent microscope . The shortening-re-lengthening features of sarcomere and the intake-discharge features of calcium were simultaneously recorded by IonOptix equipment .Results 70%rod-shaped with clear-striation adult rat cardiomyocytes could be obtained with the stated two separation medium and cultured in serum-free medium for more than 7 days.GFP could express more than 7 days when the cardiomyocytes were infected with adenovirus .Cardiomyocytes obtained by the first separation medium could not contract with the electrical stimulation, while cardiomyoctyes obtained by the second separation medium could be used for the detection of excitation -contraction coupling .The shortening fraction of sarcomere was 11.61%±2.15% and the relaxing time was ( 0.177 ± 0.031) s.The amplitude of calcium transient was 30.79% ±9.74 % and the decaying time of calcium transient was (0.300 ±0.074) s.Conclusion With the stated two separation medium , adult rat cardiomyocytes can be well isolated , cultured and infected with adenovirus .The second separation medium can be used for the detection of excitation-contraction coupling characteristics .
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The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 M L-T3, respectively. Chelation of Ca2+ using ethylene glycol-bis (2‑aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.
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Aim To observe the cardioprotection of chronic intermittent hypobaric hypoxia (CIHH) against ischemia/reperfusion injury in adult and young rats.Methods Adult and postnatal male Sprague-Dawley rats were divided randomly into four groups:control 28-day group (CON28),control 42-day group (CON42), CIHH 28-day treatment group (CIHH28), and CIHH 42-day treatment group (CIHH42). CIHH animals with maternal rats were put into a hypobaric chamber 2 days before birth to get 28 days and 42 days CIHH mimicking 3000 m altitude (P_B=525 mmHg,P_(O_2)=108.8 mmHg), 5 h daily, respectively.The control animals were kept in the same environment as CIHH rats with free access to water and food except hypoxic exposure. The isolated hearts were perfused in the Langendorff apparatus, undergoing 30 min global ischemia and 60 min reperfusion.Cardiac function was recorded continuously during the whole experiment. Parameters of left ventricular function included left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal positive (+LVdp/dt) and negative (-LVdp/dt) velocity of left ventricular pressure, coronary flow (CF) and heart rate(HR).Results ① For adult rats, there was no significant difference in the parameters of left ventricular function between CIHH28 and CON28 groups. However, the recovery of cardiac function in CIHH42 rats was much better than that in CON42, including LVDP, LVEDP, ±LVdp/dt and CF (P<0.05). ② For young rats, the basic coronary flow (CF) in CIHH rats was significant higher than that in CON rats, while other parameters of cardiac function didn't change. The recovery of cardiac function in CIHH rats was much better than that in CON rats, including LVDP, LVEDP,±LVdp/dt and CF (P<0.05).Conclusion CIHH confers cardioprotection against ischemia/reperfusion injury in rat cardiomyocytes, which is predominant in CIHH42 group and significantly affected by the age of animals. Cardioprotective effects produce more easily in young rats by CIHH.
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This study was performed to investigate the changes of immunoreactivities of calcium channel alpha(1B) subunit in myenteric plexus of capsaicin treated adult rats. Sprague Dawley rats (about 200 g) were injected with capsaicin (50 mg/kg) subcutaneously. Ileal myenteric plexus was prepared by whole mount preparation method in 1 day, 2 days, 1 week and 4 weeks after capsaicin treated adult rats. Each myenteric plexus was reacted with NADH-TR and immunostained with alpha(1B) subunit. Myenteric ganglion cell numbers were counted by image analyzer. In control group myenteric plexus was arranged in rectangular appearance; myenteric ganglia and internodal strand were immunoreacted with alpha(1B) subunit. Immunoreactive cells were 56.0% of total myenteric neurons. Total numbers of immunoreactive cells decreased by 19.0%, 37.9% and 64.9% in 1 day, 2 days, and 1 week after capsaicn treated group respectively. In 4 weeks after capsaicin treated group, immunoreactivities of alpha(1B) subunit were increased compared to that of the 1 week after group. However total numbers of immuoreactive cells decreased by 43.9% compared to that of the control group. In conclusion, we confirmed that immunoreactivities of alpha(1B) subunit were decreased until 1 week after capsaicin treatment and recovered progressively after that time. It will take more than 4 weeks latency to recover the control immunoreactivities of alpha(1B) subunit.
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Adult , Animals , Humans , Rats , Calcium Channels , Calcium , Capsaicin , Cell Count , Ganglia , Ganglion Cysts , Myenteric Plexus , Neurons , Rats, Sprague-DawleyABSTRACT
Until recently the neurotoxic effects of capsaicin to adult animals were thought to be limited to the peripheral nervous system. Several reports suggest the possibility of central nervous system changes after capsaicin administration to the adult rat. To determine the effect of capsaicin to the adult central nervous system, morphologic changes of the Lissauer tract were investigated by electron microscopy. The number of axons and substance P and CGRP immunore-active axons of the tract of Lissauer were also counted and analyzed by electron microscopic and immunocytochemical techniques. Ultrastructural degenerative changes were observed in the Lissauer tract of the capsaicin treated adult rats. The number of axons in the tract of Lissauer were decreased in most cases of the capsaicin treated rats. The number of substance P and CGRP immunoreactive axons in the tract of Lissauer were decreased in the capsaicin treated rats. The number of substance P and CGRP immunoreactive axons were decreased more than those of the total axons. Numerical changes were more pronounced in unmyelinated axons than in myelinated axons after capsaicin administration. The changes of L2 spinal tract of Lissauer were more pronounced than those of T10 tract of Lissauer. In conclusion, morphologic changes of the adult rat by capsaicin are not limited to the peripheral nervous system, but extend into the central nervous system.
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Adult , Animals , Humans , Rats , Axons , Capsaicin , Central Nervous System , Microscopy, Electron , Myelin Sheath , Peripheral Nervous System , Substance PABSTRACT
PURPOSE: Reorganization of mossy fiber terminals in the supragranular layer of the dentate has been found in hippocampi of human epileptics and animal models by Timm staining. Many studies have provided evidence that mossy fiber sprouting is strongly associated with neuronal loss. But the question of whether cell loss is necessary for stimulation of mossy fiber sprouting is remained to be answered. In this present study, we evaluated whether hippocampal mossy fiber sprouting is induced in damaged hippocampus of the rats exposed to hypoxic-ischemic insults in juvenile and adult period. METHODS: At ages of 4-5 weeks and 2 months, the experimental rats were received procedure of right carotid artery unilateral ligation under anesthesia. After 3 hours of the recovery period, they were placed in an airtight 2000ml chamber and exposed to a 8% oxygen-92% nitrogen mixture delivered at 5 liter/min for 90 minutes (juvenile) and 50 minutes (adult). After the recovery period, The animals were returned to cages and housed with controls. 2 weeks later, rats of the control and hypoxic-ischemia group were anesthetised and then perfused with sodium sulfide solusion and fixed. 40micrometer (for Timm stain) and 5micrometer (for H & E stain) coronary brain sections were obtained, stained with Timm method and H < E stain for the observation of the neuronal loss and supragranular Timm granules in the hippocampi. RESULTS: Light microscopic examination of the brains from hypoxic-ischemic animals demonstrated ischemic changes of variable degrees in the hippocampal hilar and pyramidal cell layers. No supragranular mossy fiber sprouting were found in hippocampi of juvenile and adult rats with hypoxic-ischemic damages. CONCLUSION: These results implicated that hippocampal mossy fiber sprouting is not induced in the experimental hypoxic-ischemic encephalopathy of juvenile and adult rats, although cellular loss is found in hippocampus. Neuronal loss might be not necessary for the development of mossy fiber sprouting.
Subject(s)
Adult , Animals , Humans , Rats , Anesthesia , Brain , Carotid Arteries , Hippocampus , Hypoxia-Ischemia, Brain , Ligation , Models, Animal , Mossy Fibers, Hippocampal , Neurons , Nitrogen , Pyramidal Cells , SodiumABSTRACT
10?m cytochalasin D (CD) was used to treat cardiocytes from adult human atrium and adult rat atrium and ventricle in long-term primary cultures. Durations of treatment were 0.5, 1, 6, 12, 24 and 48h. In some cultures, the medium containing CD was removed at the planned time to be replaced with the medium without CD.These dishes were then cultured for an additional 48h. Control cultures were exposed to dimethyl sulfoxide (DMSO), the vehicle used to dissolve CD. All cultured cells were first stained with rhodamine-labelled phalloidin to show F-actin and then stained with fluoreseein-labelled antitubulin to show microtubles. The freshly isolated and rounded cardiocytes did not respond to CD, while the spreading cells responded apparently. The actin filament bundles in the peripheral zone of spreading cells were cut into segments. Most segments were gathered into aggregates and granules. Some aggregates were lodged on the inner aspect of the sarcolemma. Small vacuoles were seen between the myofibrils or somewhere along the course of the myofibrils. The CD response from the atrial spreading cells, especially the human atrial spreading cells, were more obvious than that from the rat ventricular cells. Cells exposed to CD and then cultured in normal medium for 48h did not return to normal. Microtubules were not directly affected by CD, but in places where vacuolization occured they made way for vacuoles. All the control cultures made no response to DMSO. The above-mentioned results suggest that different sensitivity to CD existed in cultured adult cardiocytes between different species and that a difference also existed in the contractile machinery between the atrial culturing cells and ventricular cultureing cells of the same species.
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Objective To evaluate the effects of ginkgolide B on inducing neural stem cells differentiating into neurons. Methods A great deal of single cell clone neurospheres raised from single cell and proliferated by the technology of serum\|free culture and single cell clone.Suspensions of cell clone neurospheres were plated equably into 24 well plates and added into the 10% FBS differentiation medium containing ginkgolide B,BDNF or without any factor.Cultures were terminated at 7 and 14 days respectively.MAP\|2,neuron\|specific marker,were used to mark neurons by immunofluorescence.MAP\|2 positive neurons were observed and counted by fluorescence microscope.The area and perimeter of these positive neurons were analyzed. Results The number of MAP\|2 positive neurons in ginkgolide B group is more than that in the control group in two periods.The area and perimeter of MAP\|2 positive neurons in ginkgolide B group were markedly larger than those in the control group at 7 and 14 days after cultured,but it's less than those in the BDNF group.Conclusion\ Neural stem cells can be induced to differentiate into neurons by ginkgolide B which has the similar role to BDNF.\;[