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Objective To investigate whether paraptosis and autophagy have an effect on acute retinal ischemia-reperfusion injury (RIRI) in an experimental rat model that recapitulates features of acute hypertensive glaucoma and to explore the possible underlying mechanisms.Methods A total of 30 adult male Sprague-Dawley rats were randomly divided into RIRI group and control group.The acute RIRI model was induced with normal saline in the right eye of rats from the RIRI group by anterior chamber perfusion,while the rats in the control group left untreated.On day 1,day 3,day 7,day 28 after RIRI model establishment,the changes in morphology of retinal ganglion cells (RGCs) were observed by transmission electron microscopy (TEM),and the expression of microtubule-associated protein 1 light chain 3 (LC3) was measured by immumofluorescence methods.Results When compared with the control group,the number of cytoplasmic vacuoles predominantly derived from the progressive swelling of mitochondria and/or endoplasmic reticulum (ER) in RGCs were increased in the RIRI group from day 1 to day 28 by TEM.And ultra-structural analyses showed the double-or multiple-membrane autophagosomes were markedly accumulated in the cytoplasm of RGCs following acute RIRI.The average number of autophagic vacuoles in the cytoplasm of RGCs was 0.79 per 50 μm2 in the control group,and the average number of autophagosomes reached to a maximum on day 7 after acute RIRI at 2.29 per 50 μm2,which was statistically significant compared with the control group (P ＜ 0.05).Compared to the control group,LC3 expression in the cytoplasm of RGCs was up-regulated on day 1 after acute RIRI,which sustained throughout the experimental period.The percentage of LC3 positive cells in the retinal ganglion cell layer was 15.90％ in the control group,and the data was 46.95％ and 52.30％ on day 1 and day 28 after RIRI,respectively,both which were statistically significant compared with the normal control group (both P ＜ 0.05).Conclusion Both paraptosis and autophagy participate in death of RGCs after acute RIRI.Programmed cell death in different cells,either coexistence of multiple-cell death form or a single-cell death form,participates in the pathogenesis of acute RIRI.
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· AIM:To study whether autophagy and paraptosis were activated in retinal ganglion cells (RGCs) after acute high intraocular pressure (lOP) in an experimental rat model and to explore the possible underlying mechanisms.· METHODS:A total of 50 male Sprague-Dawley (SD)rats were randomly divided into normal control group,and 3d,1,4,8wk group after acute elevated intraocular pressure(IOP) (n =10 per group).Acute intraocular hypertension model was established by anterior chamber perfusion of normal saline in the right eye.The expression levels of microtubule-associated protein 1 light chain 3 (LC3) was measured by immumofluorescence method.To determine whether autophagy and paraptosis were activated.Retinal sections were examined by transmission electron microscopy (TEM).Autophagosomes and cytoplasmic vacuoles in the cytoplasm of RGCs were measured.· RESULTS:TEM analysis revealed that double-and multiple-membrane vacuoles containing electron-dense materials of autophagosomes were found in RGCs.The number of autophagosomes per 50μ m2 were 0.79 ± 0.43,2.14±0.36,2.29±0.47,1.57±0.51 and 1.21±0.43 in the normal control group and in acute IOP group at 3d,1wk,4wk,8wk,respectively.The number of autophagosomes markedly increased in the cytoplasm of RGCs at 3d,1wk,4wk,8wk groups than those in the normal control group (all at P＜ 0.05).LC3 positive expression was rarely detected in ganglion cell layer (GCL) in the normal control group and percentage of LC3 positive cells was 15.90％.Immumofluorescence analysis showed that the percentage of LC3 positive cells statistically increased in acute lOP groups when compared with control group (P＜0.05).The number of RGCs per 200μm in each group of acute lOP injury significantly decreased compared with the normal control group (P ＜ 0.05).Cytoplasmatic vacuolization were observed in RGCs at 3d after acute lOP injury and lasting to 8wk.TEM also revealed that a large number of cytoplasmic vacuoles were derived predominantly from the progressive swelling of mitochondria and/or endoplasmic reticulum (ER).· CONCLUSION:Autophagy and paraptosis participate in the death of RGCs under transiently elevated intraocular pressure.Different types of programmed cell death (PCD),coexistence of multiple cell death forms or a single cell death form,participates in the pathogenesis of acute elevation of intraocular pressure.
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·AIM: To investigate the role of small interference RNA interference targeted Integrin-linked kinase (ILK SiRNA) on the proliferation and apoptosis of human Tenon fibroblasts (HTFs) induced by transforming growth factor-β2(TGF-β2). · METHODS: The HTFs were identified by immunofluorescence analysis with Vimentin and keratin. HTFs with no other addiction was as normal control;H+T group:HTFs+5μ g/L TGF-β2;H+T+NC SiRNA group:HTFs+5μ g/L TGF-β2+50nmol/L negtive SiRNA; H+T+ILK SiRNA group:HTFs+5μ g/L TGF-β2+50nmol/L ILK SiRNA. The ILK SiRNA were transfected into HTFs by lipofectamine 2000, then the cells were stimulated with 5μ g/L TGF - β2. The protein expression of ILK were analyzed by Western Blot. The proliferation levels of HTFs were analyzed by CCK-8, the apoptosis of HTFs were analyzed by Hoechst 33342/PI double staining. ·RESULTS: The protein ILK were expressed in both TGF-β2treated and control groups, and TGF- β2up-regulated the expression of ILK, ILK SiRNA inhibited the protein expression of ILK(P< 0. 05). CCK- 8 analysis showed that compared with the normal control group,the cell proliferation rate of HTFs in TGF-β2treated group increased, and in ILK SiRNA group the cell proliferation rate was suppressed after exposured to ILK SiRNA for 48h (P<0.05). Hoechst 33342/PI double staining showed that there was no change on the apoptosis of TGF - β2 stimulated group (P>0.05), compared with the normal control group, however in the ILK SiRNA group, we found lots of apoptosis cells and a few of necrotic cells (P<0.05). ·CONCLUSION: The ILK SiRNA attenuates the abnormal proliferation of HTFs induced by TGF - β2, thereby enhancing the apoptosis of HTFs.
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AIM: To study in vivo the effect of androgen on mice tear film stability and Mucins expressions in corneal epithelial cells in BALB/ c mice after orchectomy. METHODS: With orchiectomy operation, we set up mice model. And serum androgen concentration of mice was detected by radioimmunoassay. Break - up time ( BUT ) of tear film was tested in the different experimental points. Mice corneal epithelia were peeled and MUC1 and MUC4 mRNA and protein levels were measured by RT-PCR and Western blot. RESULTS: The serum androgen concentration reduced to 0ng/ μ L at 1wk after orchiectomy. The BUT values were 68. 33±12. 86s, 62. 47±3. 75s, 58. 67± 10. 03s, 47. 17±7. 64s, 39. 51±3. 39s, 32. 67±3. 88s and 31. 00±2. 36s in the normal control group, sham group and in orchectomy group at 1, 2, 4, 6 and 8wk, respectively, and the BUTs were significantly shorter in the orchectomy at 2, 4, 6 and 8wk groups than those in the normal control group (all at PCONCLUSION: In vivo, androgen regulates Mucins expressions in mice corneal epithelial cells, makes BUT shorter,and influence the stability of tear film.
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<p><b>BACKGROUND</b>Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition.</p><p><b>METHODS</b>RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3.</p><p><b>RESULTS</b>CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05).</p><p><b>CONCLUSION</b>Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.</p>
Subject(s)
Animals , Rats , Apoptosis , Physiology , Caspase 3 , Metabolism , Cell Cycle , Physiology , Cell Hypoxia , Physiology , Cell Proliferation , Physiology , Cell Survival , Physiology , Cells, Cultured , Cyclin D1 , Metabolism , NF-kappa B , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Rats, Sprague-Dawley , Retina , Cell Biology , Stem Cells , Cell Biology , Triamcinolone Acetonide , PharmacologyABSTRACT
Abstract?AlM: To investigate the effect of zebularine ( Zeb ) loaded Poly ( ethylene glycol ) - block - poly ( ε -caprolactone) methyl ether ( MePEG-PCL) nanoparticles ( NPs) on the viability, attachment, and apoptosis of in vitro cultured lens epithelial cells ( LECs) .?METHODS: In vitro cultured infant human lens tissue HLE B-3 immortalized cells were distributed randomly divided into six groups. Each group was administered with free Zeb 50μmol/L ( ZebF1 group ) , 100μmol/L ( ZebF2 group) , Zeb -loaded MePEG-PCL NPs 50μmol/L ( ZebNP1 group) , Zeb -loaded MePEG-PCL NPs 100μmol/L ( ZebNP2 group) , MePEG-PCL empty NPs( NPs group) or blank medium (group C) respectively. A tetrazolium dye assay ( MTT) test and modified MTT test were performed to determine cell viability and cell attachment. DNA ladder was used to detect the cell apoptosis.?RESULTS: Determined by MTT colorimetric method:Cell proliferation rate of LECs were suppressed by all Zeb administration groups in a concentration-time dependent manner (P ZebNP1>ZebF2 (P<0. 05).?CONCLUSlON: Zeb loaded MePEG-PCL NPs had better effect on suppressing the viability and attachment of in vitro cultured LECs than the free Zeb groups , as well as enhancing the apoptosis.
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AlM:To observe the effects of double incision combined surgery of single - stab trabeculectomy and phacoemulsification. METHODS: Totally 28 cases ( 30 eyes ) with glaucoma and cataract undertook the modified combined surgery of single - stab trabeculectomy and phacoemulsification. After traditional phacoemulsification, cut the bulbar conjunctiva and Tenons capsule from the 11 o'clock to 1 o'clock, then puncture into the anterior chamber in 2mm behind the corneal limbus with 3mm tunnel knife, shaping a 3mm wide, 1/3-1/4 thickness scleral tunnel. Getting into the trabecular tunnel, bite off 3 pieces of trabecular tissue about 1mm × 1mm size. The changes in the imtraocular pressure ( lOP ) and the visual acuity before and after the surgery as well as filtering bleb ( OCT confirmed) and complications were carefully observed in 3-6mo postoperatively. RESULTS: The postoperative visual acuity in 1wk postoperatively less than 0. 1 was found in 3 eyes, from 0. 1 to 0. 3 was found in 6 eyes,from 0. 3 to 0. 6 in 13 eyes, from 0. 6 to 0. 8 in 8 eyes. One eye had malignant glaucoma, and 8 eyes had cornea edema and slightly fibrin exudation in the pupil area; ln all cases maintained function conjunctival blebs of filtering, OCT confirmed this. lOP remained normal in 28 eyes in 3-6mo follow up, lOP of 2 other eyes could be controlled by anti-glaucoma eye drops. CONCLUSlON:Double incision combined surgery of single- stab trabeculectomy and phacoemulsification is effective and safe, reduces the postoperative complications and is worthy of promotion.
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Background Neurofilament 200 (NF200) is an indirect indicator of the specific distribution of axons.It reflects the condition of the maintenance of neuronal morphology.Whether NF200 is involved in the damage of the visual pathway after retinal ischemia reperfusion injury (RIRI) should be further examined.Objective The present study was to investigate the expression of NF200 in retinal ganglion cells (RGCs),lateral geniculate nucleus (LGN),superior colliculus and visual cortex following RIRI.Methods Forty SD rats were randomized into the RIRI 1-,2-,3-,4-,6-,8-week groups,sham operation group and control group.Acute intraocular hypertension was induced in the right eye by anterior chamber perfusion of normal saline solution for 60 minutes to elevate the intraocular pressure to 110 mmHg.The animals were sacrificed at different time points for the preparation of the retina,LGN,superior colliculus and visual cortex sections.The expression of NF200 in RGCs,LGN,superior colliculus and visual cortex was assayed by immunohistochemistry.Results The expression level (A value) of NF200 in the RGCs was significantly different among the 8 groups after reperfusion (F =78.855,P =0.000),and that in the 1-week group was significantly lower than in the control group (t =36.563,P＜0.01).In the RIRI 1-week group,the expression of NF200 in the contralateral LGN in the experimental group was significantly lower than that in the control group (t =6.483,P＜0.01).In the 4-week group and 6-week group,the expression of NF200 in the contralateral LGN was significantly higher than that in the control group (t =2.904,4.313,P＜0.01).One week after RIRI,the expression of NF200 in contralateral superior celliculus in the experimental group was significantly lower than that in the control group (t =2.966,P＜0.05),and in the 2-week group,the expression of NF200 in the contralateral superior colliculus was significantly higher than that in the control group (t =7.397,P＜0.01).In the 2-week group,3-week group and 4-week group,the expression of NF200 in bilateral visual cortex was significantly higher in the experimental group than that in the control group (contralateral ∶ t =18.728,18.213,15.088,P＜0.01 ; ipsilateral ∶ t =8.690,5.704,7.805,P＜0.01).Conclusions RIRI can induce axonal damage of RGCs,contralateral LGN,superior colliculus and bilateral visual cortex neurons.
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<p><b>OBJECTIVE</b>To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples.</p><p><b>METHODS</b>The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells).</p><p><b>RESULTS</b>When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples.</p><p><b>CONCLUSION</b>A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.</p>
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Animals , Male , Rats , Capillaries , Pathology , Cerebral Cortex , Pathology , Lasers , Microdissection , Methods , Neurons , Pathology , RNA , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers.</p><p><b>METHODS</b>EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy.</p><p><b>RESULTS</b>After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture.</p><p><b>CONCLUSION</b>we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.</p>
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Humans , Cell Hypoxia , Enhancer Elements, Genetic , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins , Genetics , HeLa Cells , Microscopy, Fluorescence , Plasmids , TransfectionABSTRACT
AIM: To investigate the clinical effectiveness of reformed nonperforating trabeculectomy (NPT) in the patients with primary glaucoma.METHODS: 30 eyes of 21 patients with primary glaucoma patients underwent reformed NPT, in which 1.0mm3.0mm out layer of trabecular meshwork with Schlemm's canal was excised, but the innermost tissue was reserved. The clinical effectiveness was observed with short-term follow-up (1wk;1,6mo) and the long-term follow-up (1a and over, 10a the longest).RESULTS: The intraocular pressure (IOP) was controlled in 28 eyes (93%) without anti-glaucoma medicine during short-term follow-up, and in 27 eyes (90%)for long-term ones. There were no serious post-operative complications in all cases.CONCLUSION: The elevated IOP in patients with primary glaucoma can be effectively reduced by reformed NPT during short-term and long-term follow-up. The post-operative complications are much less because of no intra-operative penetration of the anterior chamber. The reformed NPT is an ideal surgical procedure for primary glaucoma.
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<p><b>OBJECTIVE</b>To investigate histamine-induced changes of the intracortical vessels in the cortical slice of rat brain.</p><p><b>METHODS</b>Immunohistochemistry was employed to detect the expression of H1 and H2 receptors in the intracortical blood vessels of rats. Histamine-induced constriction of the intracortical blood vessels of the brain slices was observed with differential interference contrast microscope. Measurements of the luminal diameter were made on-line during the course of the experiment and confirmed off-line from the stored images. In order to observe whether histamine H1 and H2 receptors affected histamine-induced constriction, the intracortical blood vessels in the brain slices were pre-treated with H1 receptor antagonist diphenhydramine and H2 receptor antagonist cimetidine.</p><p><b>RESULTS</b>Expression of H1 and H2 receptors was detected in the intracortical blood vessels of the rat brain. Histamine (1-100 micromol/L) induced a concentration-dependent constriction from (1.48-/+0.67)% to (32.91-/+7.91)%. The reactions to each histamine concentration were significantly (P<0.01) different from each other, with the exception of the highest histamine concentrations (30 and 100 micromol/L) when maximal constriction due to histamine were observed (P>0.05). With pre-treatment of the slice with 10 micromol/L diphenhydramine, application of histamine did not elicit constriction. Pre-treatment of the slice with 10 micromol/L cimetidine did not completely inhibit but somehow significantly weakened vascular constriction in response to histamine treatment at 10 and 30 micromol/L (P<0.05).</p><p><b>CONCLUSION</b>Histamine can induce constriction of the intracortical blood vessels, which is mediated by H1 receptor.</p>
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Animals , Male , Rats , Blood Vessels , Metabolism , Physiology , Cerebral Cortex , Cimetidine , Pharmacology , Diphenhydramine , Pharmacology , Histamine , Pharmacology , Histamine H1 Antagonists , Pharmacology , Histamine H2 Antagonists , Pharmacology , In Vitro Techniques , Rats, Sprague-Dawley , Receptors, Histamine H1 , Metabolism , Physiology , Receptors, Histamine H2 , Metabolism , Physiology , VasoconstrictionABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of citicoline on spatial learning and memory of rats after focal cerebral ischemia.</p><p><b>METHODS</b>The rats were randomly divided into sham-operation group, ischemia control group and citicoline group. In the later two groups, focal cerebral ischemia model was established by introducing an intraluminal filament into the left middle cerebral artery, and citicoline (500 mg/kg) or 0.9% NaCl was administered intraperitoneally once a day for 2 weeks after the operation. The rats in the sham-operation group were not subjected to middle cerebral artery occlusion (MCAO) with intraluminal filament. The spatial learning and memory functions of the rats were evaluated by Morris water maze test 15 days after MCAO for 5 days.</p><p><b>RESULTS</b>The rats in ischemia control group exhibited serious spatial learning and memory deficits in both place navigation test and spatial probe test. In the former test, the mean escape latency of citicoline-treated rats were significantly shorter than that of ischemia control rats (P<0.01), and in the latter test significant diffidence was noted between citicoline and ischemia control groups in the percentage time spent in the former platform quadrant and frequency of crossing the former platform (P<0.05).</p><p><b>CONCLUSION</b>Citicoline can improve the spatial learning and memory function of rats after focal cerebral ischemia.</p>