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Hypoxia is beneficial for the differentiation of stem cells transplanted for myocardial injury, but mechanisms underlying this benefit remain unsolved. Here, we report the impact of hypoxia-induced Jagged1 expression in cardiomyocytes (CMs) for driving the differentiation of cardiac stem cells (CSCs). Forced hypoxia-inducible factor 1 (HIF-1) expression and physical hypoxia (5% O) treatment could induce Jagged1 expression in neonatal rat CMs. Pharmacological inhibition of HIF-1 by YC-1 attenuated hypoxia-promoted Jagged1 expression in CMs. An ERK inhibitor (PD98059), but not inhibitors of JNK (SP600125), Notch (DAPT), NF-B (PTDC), JAK (AG490), or STAT3 (Stattic) suppressed hypoxia-induced Jagged1 protein expression in CMs. c-Kit CSCs isolated from neonatal rat hearts using a magnetic-activated cell sorting method expressed GATA4, SM22 or vWF, but not Nkx2.5 and cTnI. Moreover, 87.3% of freshly isolated CSCs displayed Notch1 receptor expression. Direct co-culture of CMs with BrdU-labeled CSCs enhanced CSCs differentiation, as evidenced by an increased number of BrdU/Nkx2.5 cells, while intermittent hypoxia for 21 days promoted co-culture-triggered differentiation of CSCs into CM-like cells. Notably, YC-1 and DAPT attenuated hypoxia-induced differentiation. Our results suggest that hypoxia induces Jagged1 expression in CMs primarily through ERK signaling, and facilitates early cardiac lineage differentiation of CSCs in CM/CSC co-cultures HIF-1/Jagged1/Notch signaling.
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Objectives To explore the effect of exogenous erythropoietin (EPO) on the expression of glial fibrillary acidic protein (GFAP) in hippocampal CA1 region and 5- bromide -2- uracil (BrdU) in hippocampal DG region in neonatal Wistar rats with hypoxic-ischemic brain damage (HIBD). Methods Forty-eight Wistar rats aged 7 days were randomly divided into HIBD model group and EPO experimental group, and another 24 rats as sham operated group. The HIBD model was established by ligating the right common carotid artery and inhaling hypoxia gas mixture (8％ O2 and 92％ N2) for 2 h. The expression of GFAP in hippocampal CA1 region and the number of BrdU positive cells in the hippocampus were detected by immunohistochemical method on at 14 d, 21 d, and 28 d after operation and compared among three groups. Results On 14 d and 21 d after operation, the expression of GFAP in CA1 region and the number of BrdU positive cells were statistically different among three groups (P<0.01) with EPO experimental group having the highest, HIBD model group having the second highest and sham operation group having the lowest in both, . On 28 d after operation, there was no difference in the expression of GFAP and the number of BrdU positive cells in the DG among three groups (P>0.05). At different time point (14 d, 21 d, 28 d) in every group, the expression of GFAP in CA1 region and the number of BrdU positive cells in DG region were all statistically different (P<0.01), all with the highest on 14 d after operation, second highest on 21 d, and the lowest on 28 d. Conclusions Early administration of exogenous EPO can promote the expression of GFAP in hippocampal CA1 region and increase the number of BrdU positive cells in DG region, which indicates that EPO can promote the proliferation and regeneration of damaged neurons. EPO had neuroprotective effect on neonatal rats with hypoxic-ischemic brain damage.
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This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats.The behavior of rats with experimental chronic neuropathic pain was observed,and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected.Thirty-two male Sprague-Dawley rats were randomly divided into four groups:sham-operated group,sham-rTMS group,1 Hz group and 20 Hz group (8 rats in each group).Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group.Then we applied different frequencies of rTMS to the primary motor cortex (M1) contralateral to the pain side once daily for 10 consecutive days.Pain behavior scores were observed before and after treatment.Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs.Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2-deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn.After rTMS treatment,the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P＜0.05).Moreover,the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P＜0.05),suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated.Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.01) after rTMS treatment.Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.After rTMS treatment,the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.05).In addition,the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P＜0.05).It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.
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This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats.The behavior of rats with experimental chronic neuropathic pain was observed,and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected.Thirty-two male Sprague-Dawley rats were randomly divided into four groups:sham-operated group,sham-rTMS group,1 Hz group and 20 Hz group (8 rats in each group).Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group.Then we applied different frequencies of rTMS to the primary motor cortex (M1) contralateral to the pain side once daily for 10 consecutive days.Pain behavior scores were observed before and after treatment.Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs.Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2-deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn.After rTMS treatment,the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P＜0.05).Moreover,the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P＜0.05),suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated.Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.01) after rTMS treatment.Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.After rTMS treatment,the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.05).In addition,the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P＜0.05).It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.
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ObjectiveTo investigate the effects of caffeine citrate (CC) on the neuronal proliferation and apoptosis and long-term learning ability in neonatal rats with hypoxia-ischemic brain damage (HIBD).MethodsForty-eight 7-day-old Sprague-Dawley neonatal rats were randomly divided into 3 groups: sham operation group (n=16), HIBD group (n=16), HIBD + caffeine citrate group (CC group,n=16). Rats in HIBD and CC groups received ligation of left common carotid artery followed by 2 hours of hypoxia to establish HIBD model. Rats in CC group were injected intraperitoneally with CC (20 mg/kg) before and at 0 min, 24 h, 48 h, and 72 h after hypoxia-ischemic (HI), and rats in the other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time. Meanwhile, from postnatal day 10, each rat was injected intraperitoneally with 5-bromo-2’-de-oxyuridine (BrdU) (50 mg/kg) for 5 consecutive times, once every 12 h. On postnatal day 12, BrdU in the hippocampal dentate gyrus and cleaved caspase-3 in the hippocampal CA1 area were detected by immunohistochemistry, and neuronal apoptosis in hippocampal CA1 area were detected by TUNEL staining. On postnatal day 28, long-term learning and memory ability of rats was tested by Y maze.ResultsThere was signiifcant difference in the number of BrdU-positive cells in brain tissues of rats among three groups (F=101.38,P<0.01). The BrdU-positive cells in HIBD group and CC group were signiifcantly more than those in sham operation group (P<0.05). There was signiifcant difference in the number of cleaved caspase-3-positive cells in hippocampal CA1 area among three groups (F=379.77,P<0.01). The cleaved caspase-3-positive cells in CC group were signiif-cantly fewer than those in HIBD group but signiifcantly more than those in sham operation group (P<0.05). The TUNEL-pos-itive cells in hippocampal CA1 area were signiifcantly different among three groups (F=505.92,P<0.01) which was most in HIBD group and fewest in sham operation group and signiifcant difference was found through multiple comparison (P<0.05). The total learning number of avoiding electric shock tested by Y maze was signiifcantly different among three groups (F=32.05, P<0.01) which was most in HIBD group. Correct response rate was significantly different among three groups (F=24.99, P<0.01) which was lowest in HIBD group.ConclusionsCaffeine citrate can improve the ability of long-term learning and memory in neonatal rats with hypoxia-ischemic brain damage, the mechanism of which may be related to reducing the neuronal apoptosis after hypoxia ischemia.
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The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5.There was significant difference between the experimental group and the control group (P＜0.001).Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.
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Purpose: The goal of this study is to define whether or not preoperative portal vein embolization has any additional role in the total amounts of liver regeneration and functional improvement after major hepatectomy in rat model. In addition, this study is to define obstructive jaundice has any positive or negative effect on it. METHODS: There were a total of 650 rats, divided into three experimental groups. Experiment A was done under the normal liver status, experiment B was done under the obstructive jaundice status, experiment C was done under the external biliary drainaged status. Each experimental group was divided into three groups that had been made by different surgery. One was 70% partial hepatectomy, another was 70% portal vein branch ligation, and the other was 70% portal vein ligation followed by 70% hepatectomy. Each operational group required over 60 rats for serial data collection which was taken at the operation and 6, 12, 24, 48, 72 hours after operation. RESULTS: We finally observed that there was no additional regeneration of remaining liver by doing preoperative portal vein embolization. It was same in obstructive jaundice group and external biliary drainaged group. And also, there was no significant fucntional improvement or deterioration by existence of obstructive jaundice. Conclusion: We conclude it is no worth doing preoperative portal vein embolization for getting additional liver regeneration and obstructive jaundice does not has significant positive or negative effect on liver regeneration and hepatic function in itself.
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Animals , Rats , Data Collection , Hepatectomy , Jaundice , Jaundice, Obstructive , Ligation , Liver Regeneration , Liver , Models, Animal , Portal Vein , RegenerationABSTRACT
Objective:The purpose of this study is to investigate the feasibility of labeling skin of inner mongolia cashmere goats with BrdU,and to optimize its immunohistochemical conditions.Methods:We injected different dose of BrdU into the neck vein of cashmere goats,then we took samples from the back skin of the goats,fixed in 4% paraformaldehyde.Next the samples were embedded with paraffin and sectioned.We examined the samples with immunohistochemical technology to test the effect of the influencing factors to labeling results as the BrdU injection,such as dosage,the concentration of second antibody and streptavidin-peroxidase,the way of antigen pretreatment,and DNA denature temperature.Results:We observed red BrdU positive cells in labeled goats whereas not in the control.Meanwhile,in the samples of the unlabeled goats or those incubated with PBS,no nuclear staining is observed.Conclusion:BrdU can be used for labeling of inner mongolia cashmere goats in vivo,which have no obvious dosage effect.The best condition of labeling is that puttig the section in 2 mol/L HCl 30 min at 37℃,in trypsin 30 min at 37℃.The most appropriate concentration of biotin-conjugated second antibody and streptavidin-peroxidase is 1∶100,under which the results of immunohistochemical are the best.
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BACKGROUND AND OBJECTIVES: Follicular cells of the thyroid gland has been considered an important factor affecting the thyroid gland regeneration.[(3)H]Thymidine autoradiography and immunohistochemical staining with BrdU have been commonly used and are reliable techniques for examining cell proliferation. However, these methods are not suitable for the routine analysis of cell proliferation kinetics, because they are fraught with problems such as needing fresh tissues and handling radioactive or toxic susbstances used as markers. The purpose of this study is to investigate the utility of immunohistochemical staining with a monoclonal antibody to PCNA during the healing process of a rat thyroid gland. We also compared this method with the immunohistochemical staining using a monoclonal antibody to BrdU. MATERIALS AND METHODS: We investigated the proliferative activity in regeneration of the partially resected rat thyroid tissue by using immunohistochemical stainings of PCNA and 5-bromo-2'-deoxyuridine (BrdU). The two methods were compared. RESULTS: Cell proliferative activity of regenerated follicular cells around the resected margin showed a continuous acceleration for 72 hours, and the PCNA-labeled cells stained the nuclei as clearly discernible as those of the BrdU-labeled cells. In addition, the immunohistochemical staining of PCNA provided reproducible and quantifiable results without the requirement of pretreatment. CONCLUSION: Immunohistochemical staining of PCNA and BrdU may represent as a useful technique for analysis of proliferative activity during regeneration of the thyroid glands in rats.