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Background Diabetic retinopathy (DR) is one of the most important microvascular complications of diabetes,which has become one of the leading causes of blindness.Neovascularization is the main pathological manifestations of DR,but its mechanism is unknown.There is a clear need to investigate its pathogenesis which can offer potential therapeutic targets.Objective The aim of this study was to investigate the expression and distribution of visfatin and vascular endothelial growth factor (VEGF) in diabetic model rats.Methods This study was approved by Animal Ethic Committee of Inner Mongolia Medical University.Sixty SPF 8-week-old male SpragueDawley rats were randomized into the diabetic group and control group.The rats were housed under a condition that alternated between 12 hours of light and darkness,with free access to rat food and water.Diabetes was induced by intraperitoneal injection of 60 mg/kg (0.60 ml/100 g) of streptozotocin (STZ) and control rats received equivalent volume of buffer.The models were regarded as successful when blood glucose was ≥ 16.7 mmol/L.Rats were sacrificed 12 weeks after the injection of STZ and retinal specimens were prepared to detect the expression of visfatin and VEGF.Total retinal protein was isolated from the retinas of experimental and control eyes,and the expression of visfatin and VEGF was assessed by Western blot.Frozen cross sections of retinas of 5 μm thickness were used to perform double immunofluorescence staining with anti-visfatin and anti-VEGF antibodies.Results Mean body weight of the diabetic rats was (189.02±11.34) g and that of the control rats was (489.57 ± 14.48) g at 12 weeks post-injection,showing a significant difference between them (t =5.236,P =0.003).Mean blood glucose level was (29.25±3.86) mmol/L in the diabetic group and (5.32±1.01) mmol/L in the control group,demonstrating a significant difference (t =11.778,P =0.000).Double immunofluorescence staining showed reduced expression of visfatin and VEGF in the retinal nerve fibrous layer and glial cells in the control rats.A stronger staining for visfatin and VEGF was found in the various layers of the retina in the diabetic rats,with an expression level of visfatin (A value) of 346.26±41.23,which was considerably higher than that of the control group (102.07±65.01) (t =8.291,P =0.000) in 12 weeks after injection.Furthermore,the expression of VEGF in the retina was elevated in the diabetic group compared with the control group (A value) (415.88±92.15 vs.113.06±32.06) (t=10.067,P=0.000).Conclusions Visfatin might contribute to the pathologic progression of diabetic retinal,neovascularization and it might play a synergistic role with VEGF in the pathophysiology of DR.
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Objective To investigate the clinical and immune pathological features of Ullrich congenital muscular dystrophy (UCMD) with sarcolemma-specific collagen Ⅵ deficiency (SSCD). Methods The clinical aspects of 2 patients with SSCD were analyzed and the muscle specimens from them were studied by immunofluorescence. Results SSCD patients were clinically characterized by neonatal hypotonia with proximal contractures and distal hyperlaxity at birth or early infancy. Immunofluorescence staining revealed partial deficiency of collagen Ⅵ. Double immunofluorescence staining revealed sarcolemma-specific deficiency of collagen Ⅵ, while collagen Ⅳ intact in thesarcolemma. Conclusions The clinical picture and severity of UCMD with SSCD are similar to the cases with collagen Ⅵ complete deficiency. The proximal contractures and distal hyperlaxity are the clinical hallmarks of both types. Sarcolemma-specific collagen Ⅵ deficiency can be better demonstrated by double immunofluorescence staining.
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Objective To investigate the changes of the expression of GDNF and GDNFR?1 of long-term denervation of posterior cricoarytenoid muscles(PCAMs).Methods 38 patients with vocal paralysis were grouped into four according to their denervated period of time while 12 normal PCAMs as control group.Using double immunofluorescence stain,changes of GDNF and GDNFR1 expression were observed in myofibers at different time points after denervation.Results Double immunofluorescence stain with antibodies against GDNF and GDNFR1 showed no staining in the control group,and study groups.However,after the muscle denervation lasted for 6-12 months and 1-2 years,noted was a significant accumulation of GDNF and GDNFR1 protein in cytolemma and endochylema of myofiber.The mean grey scales and positive region ratios were compared using the image analysis system.The results revealed the levels of GDNF and GDNFR1 protein expression in 6-12 months group,1-2yr group changed significantly(P0.05).Conclusion The changes in expression of GDNF and the acceptor GDNFR-?1,a powerful neurotrophic factor,implied that a good nervous regenerated microenvironment in PCAMs within 2 years.This experiment indicated that denervated posterior cricoarytenoid muscles are able to regain their functions through reinneration within 2 years.
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Widespread brain-derived neurotrophic factor (BDNF) mRNA expression has been detected in the region of catecholamine groups of the rat lower brainstem, while few BDNF-immunoreactive cells were found in this area. In the present study, a double-color immunofluorescence (IF) technique for BDNF and tyrosine hydroxylase (TH) after colchicine treatment was employed to evaluate the possible presence of BDNF immunoreactivity in the catecholamin-ergic cells of rat lower brainstem. Additionally, a double-color IF technique for BNDF and TH and in situ hybridiza-tion for BDNF mRNA were performed to see effects of hemorrhage on the expression of BDNF and its mRNA. We detected many new BDNF-immunoreactive cells in the A1, A2, A4, A6-A10 and C1-C3 cell groups and in the other lower brainstem nuclei where, without colchicine treatment, BDNF mRNA was expressed, but not BDNF immunoreactivity. In addition, the catecholaminergic neurons were found to express BDNF immunoreactivity with the co-existence being greatest, in percentage terms, in medullary catecholaminergic cell groups. Hypotensive hemorrhage, which activates medullary catecholaminergic neurons, induced the expression of BDNF immunoreactivity in catecholaminergic neurons (A1/C1 and C2) and increased the number of BDNF mRNA-containing neurons in the area. These results demonstrate that BDNF is regulated by activity in medullary catecholaminergic cell groups involved in central cardiovascular regulation.
Subject(s)
Animals , Rats , Brain Stem , Brain-Derived Neurotrophic Factor , Colchicine , Fluorescent Antibody Technique , Hemorrhage , In Situ Hybridization , Neurons , RNA, Messenger , Tyrosine 3-MonooxygenaseABSTRACT
This study aimed to carry out the reconstruction of whole tract of the vagus nerve using new powerful neurotracer which can migrate easily to the neighboring neurons through synapse and identify whether catecholaminergic neurons exist or not in the central vagal pathways. Pesudorabies virus (PRV-Ba) was used as a neurotracer and antibody to the PRV-Ba was used to localize the tracer in neurons immunohistochemically. The PRV-Ba was injected into the cervical portion of the vagus nerve of Sprague-Dawley rats. After 3 to 4 days of survival periods, brain tissues were fixed, sectioned and stained using anti-PRV-Ba and ABC method subsequently. Motor neurons of the vagus nerve were originated exclsively from dorsal motor nucleus of the vagus nerve and nucleus ambiguus in the medulla oblongata which project fiber by way of nucleus tractus solitarius up to the cerebrum including the paraventricular nucleus. Double labelled neurons were found mostly throughout the brainstem. The adrenergic inputs arose from the C1, C2, and C3 cell groups. Noradrenergic inputs originated predominately from A5 cell group, with lesser contributions from A1 and A7 cell groups as well as locus ceruleus. Some weakly stained TH-immunoreac-tive neurons, presumably dopaminergic, were labelled in the paraventicular nucleus. In conclusion, motor neurons projecting to the vagus nerve includes noradrenergic neurons of the brainstem and from a dopaminergic neurons in the paraventicular nucleus.
Subject(s)
Animals , Rats , Adrenergic Neurons , Brain , Brain Stem , Cerebrum , Dopaminergic Neurons , Herpesvirus 1, Suid , Locus Coeruleus , Medulla Oblongata , Motor Neurons , Neurons , Paraventricular Hypothalamic Nucleus , Rats, Sprague-Dawley , Solitary Nucleus , Synapses , Vagus NerveABSTRACT
Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR,PCR products cloning into PCR2.1TOPO vector,Cx36-pcDNA3 expression vector construction,cell culture,transient transfection using lipofectamine 2000,stable clone screening with G418,Western blotting,double immunofluorescence,and immunoprecipitation(IP) were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells.Using homogenates from Cx36 transfected HeLa cells,Western blotting showed Cx36 band.By immunofluorescence microscopy,Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells.Irmmunolabeling for ZO-1 in these cells exhibited similar distribution to Cx36.By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact.Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody,blots of IP protein probed with Cx36 antibody showed detectin of Cx36.Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells.Conclusion Cx36 was expressed in Cx36 transfected HeLa cells,Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.
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Objective Applying a reliable precise method to assess the mitotic index of cardiomyocytes,to disclose of disclosure the mechanism implicated in cardiomyocytes proliferation. Methods H9c2(2-1) cardiomyocytes were originally developed from rat BD1X heart(ATCC).These cells were cultured on coverslips.Double immunofluorescence staining with monoclonal antibodies(1:100) against phospho-histone H3 and ?-sarcomeric actin was performed on the cultured cells.Anti-mouse IgG FITC was used as the secondary antibody for the H3P antibody,and anti-mouse IgM Cy3 was used as the secondary antibody for the ?-sarcomeric actin.DNA was visualized with Hochest 33342.All photographs were taken with an Olympus fluorescence microscope. Results The cytoplasm of cardiomyocytes appeared red,the mitotic chromosomes green with distinct shape,and Hochest 33342-stained nuclei blue.Conclusion Our method is the reliable and exact means to observe and assess cardiomyocytes mitosis.
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Objective To investigate the expression of endometrial developing-related genes,the endometrium-like structure and cells during spontaneous differentiation of hESCs. Methods (1) Embryoid bodies were cultured in suspension for 14 days,fixed in 10% neutral-buffered formalin and embedded in paraffin.Estrogen receptor(ER) was detected by immunocytochemical staining.(2) hESCs were cultured in a 35 mm plastic dish and differentiated spontaneously for 10 days.The expression of ER and Vimentin/Keratin was detected by double immunofluorescence histochemistry.(3) hESCs were cultured in a 35 mm plastic dish and differentiated for 5 days.The expressions of endometrial developing-related genes including Wnt4,Wnt7a,Wnt5a,Hoxa10,Hoxa11 and ER were detected by RT-PCR. Results (1) The endometrium-like structures were detected in 14-day-old EBs and some were positive for ER staining.(2) Some spontaneously differentiated cells from hESCs for 10 days were positive for ER together with Vimentin/Keratin.(3)Wnt7a,Wnt4,Hoxa10,Hoxa11 and ER were detected by RT-PCR in hESCs which were differentiated spontaneously for 5 days.Conclusion During the spontaneous differentiation of hESCs,some cells are liable to differentiate into endometrial stem cells and endometrium-like cells.However,further identifications of the whole development process are needed to be done in the future.