In the present study the specific and nonspecific cholinesterase activities of the rabbit's retinae in the fetus, the neonatal, the light-isolated, and the reopened group, which consisted of 65 healthy young rabbits, weighing about 300 to 500 gm, 33 rabbit's fetuses, and neonatal rabbits, were histochemically ovserved by means of the cholinesterase method recommended by Gerebtzoff (1953) and the embedding and sectioning method pesented by Koelle and Friedenwald (1950). Cholinesterase activity of the retinae in the 15 days fetuses was not present but began to develop in the 20 days fetuses. In the 1 week group after suturing the eyelids, the most remarkable activity of specific and nonspecific cholinesterase was observed in the posterior polar area. The nearer to the peripheral area of the retina the weaker the enzymetic activities became. In the 2 weeks group after suturing eyelids, the enzymatic activity was reduced. In the 4, and 8weeks groups after suturing the eyelides, the enzymatic activities were remarkably reduced. In the l4 days after reopening eyelide, which group has previously been kept under the condition of light isolation for 4 weeks, enzymatic activities were fairly recovered and compared with the normal control group. Consequently it is histochemically deduced that the gradual change of specific cholinesterase activities in the rabbit's retinae was closely related to the visual function.
Animals ; Animals, Newborn/enzymology ; Cholinesterases/*metabolism ; Histocytochemistry ; *Rabbits ; Retina/embryology/*enzymology

In this work, the experimental conditions for two-dimensional gel electrophoresis of subretinal fluids (SRF) matrix metalloproteinases were established. The conditions tested included the composition of lysis solution and lysis method, the composition of rehydration solution and isoelectric focusing program (IEF), the composition of equilibration buffer and equilibration process and the composition of incubation solution and incubation methods. The main equipments used were IPGphor isoelectric focusing system from Amersham pharmacia and PROTEAN II xi cell from Bio-Rad, the gel strips used were the 18 cm long, pH 3 - 8 Linear immobiline DryStrips. Among the 9 samples analyzed, 2 were PVR-A, 2 were PVR-C1, 2 were PVR-C2, 2 were PVR-C3 and the remaining one could not be classified definitely. The new 2-DE MMPs method is better than Gelatin SDS-PAGE zymograhpy method, as it is higher in resolution, sensitivity and reproducibility. The experimental results suggested that the four types of MMPs expressed differently at different stages of PVR. Two of the MMPs isomers have same molecular weight (MW) but different in isoelectic points (pI). The four MMPs are determined to be MMP-1, MMP-2, MMP-9 and MMP-9, with MMP-9 has two active forms. In addition, MMP-9 and MMP-1 may be present in PVR-A samples but not in PVR-C samples, whereas MMP-2 is present in PVR-C but not in PVR-A samples. These results revealed the complex profiles of MMPs' expression in PVR. The new method can be applied to test MMPs expression in tissues, cells and other types of samples with a little modification in the protocol, and can be followed by mass spectroscopic analysis of MMPs.
Electrophoresis, Gel, Two-Dimensional ; Humans ; Isoelectric Focusing ; Matrix Metalloproteinases ; analysis ; Reproducibility of Results ; Retina ; enzymology

Group totalling 55 young rabbits (both sexes), whose right optic nerves had been severed intraorbitally, were fed for 1 week, 2 weeks, 4 weeks and 8 weeks respectively. The retina of the left eye was used as a control and that of the right eye for the experiment. The histochemical changes of cholinesterase, acid phosphatase and ribonucleic acid in the reitna after to severance of the optic nerve were observed for 8 weeks after section. In the retina of the young rabbit, whose visual connection to the central nervous system was blocked, there was a decreasing specific cholinesterase activity beginning at the 4th week after the section of it. By the 8th week, the enzyme activity in the perikaryon of the ganglion cell and the inner plexiform layer was considerably decreased. Acid phosphatase activity in the young rabbit's retina peaked at the 2nd week, but decreaseed below normal after the 4th week. This rapid decline of acid phosphatase activity was characteristic in the experimental retinae and was in contrast to the rather slow alteration of enzymatic activity in neurons undergoing wallerian degeneration. Pyroninophilic granules contained in neural cytoplasm of the retina were affected by the surgical blocking of the visual connection with the central nervous system. By the 4th week the granules had partially disappeared from the perikaryon of the ganglion cell and from the inner nuclear layer. Consequently, as the result of histochemical studies, firstly it is postulated that the gradual decline of specific cholinesterase activity in the rabbit's retina was closely related to the intraorbital blocking of the optic nerve, and secondly, that the typical degeneration of the ganglion cell in the ganglion cell layer (which was associated with a partial disappearance of the ganglion cell) was related to the changes in the acid phosphatase activity and alteration of the pyroninophilic granules in the retina following optic nerve transection.
Acid Phosphatase/metabolism* ; Animal ; Cholinesterases/metabolism* ; Histocytochemistry ; Nerve Degeneration ; Neurons/enzymology ; Optic Nerve/surgery* ; Rabbits ; Retina/enzymology* ; Substances: ; Cholinesterases ; Acid Phosphatase

OBJECTIVETo investigate the effect of carbon disulfide (CS2) on inducible nitric oxide synthase (iNOS) and apoptosis in in the retina of rats.

METHODSTwenty-four male SD rats were randomly divided into 3 groups the control group, the group receiving high-dose CS2 and the group receiving low-dose CS2. After treatment, retina were harvested and made into slice. The thickness of inner retina including outer plexiform layer, inner nuclear layer, ganglion cell layer, optic nerve fiber and inner limiting membrane was measured. Expression of iNOS in retina was measured by NADPH-NDP histochemical assay. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).

RESULTS(1) The thickness of inner retina decreased in groups receiving CS2. There was significant difference between control group and groups receiving CS2 (P < 0.05). There was also significant difference between group receiving high-dose CS2 and group receiving low-dose CS2 (P < 0.05). (2) The expression of iNOS increased in groups receiving CS2. There was significant difference between control group and groups receiving CS2 (P < 0.05 or P < 0.01). There was also significant difference between the group receiving high-dose CS2 and the group receiving low-dose CS2 (P < 0.05). (3) Apoptosis was observed in groups receiving CS2. There were significant differences in apoptosis index between the control group and groups receiving CS2 (P < 0.05 or P < 0.01). There was also significant difference between the group receiving high-dose CS2 and the group receiving low-dose CS2 (P < 0.05).

CONCLUSIONSCS2 can evoke the expression of iNOS, and the nitric oxide thus produced can be one of the causes of retina destruction. And apoptosis is also one of the causes of retina destruction.

Animals ; Apoptosis ; drug effects ; Carbon Disulfide ; toxicity ; Male ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; enzymology ; pathology

BACKGROUNDDiabetes-related pathogenic factors can cause retinal ganglion cell (RGC) apoptosis, but the specific mechanism is not very clear. The aim of this study is to investigate the correlation between glycogen synthase kinase-3 (GSK-3) activation and retinal neuron apoptosis.

METHODSIn an in vitro experiment, the number of apoptotic RGC-5 cells differentiated by staurosporine was evaluated via flow cytometry and nuclei staining using Hoechst 33258. GSK-3 phosphorylation and caspase-3 activation in RGC-5 cells after serum deprivation were determined using Western blotting. Mitochondrial membrane potential was detected using the dye 5, 5', 6, 6'tetrachloro-1, 1', 3, 3'-tetrethyl benzimidalyl carbocyanine iodide, and reactive oxygen species (ROS) levels were measured with dihydroethidium. In an in vivo experiment, the number of apoptotic retinal neurons was evaluated via terminal transferase dUTP nick-end labeling (TUNEL), and GSK-3 phosphorylation was determined using Western blotting, in the retinal nerve epithelial tissue of rats in which diabetes was induced by intravenous tail-vein injection of streptozotocin for 4 weeks.

RESULTSThe levels of phosphorylated Ser21/9 in GSK-3α/β and p-T308/S473-AKT were lower and the cleaved caspase-3 levels were higher in the serum-deprived model (P < 0.05). Lithium chloride treatment was associated with a slower rate of apoptosis, increased mitochondrial membrane potential, and decreased ROS levels in differentiated RGC-5 cells (P < 0.05). The level of blood glucose and the number of TUNEL-positive cells in the whole-mounted retinas were higher (P < 0.01), and the levels of phosphorylated Ser21/9 in GSK-3α/β and body weight were lower (P < 0.05). However, the thickness of the retinal nerve epithelial layer was not significantly less in diabetic rats compared with control group. Lithium chloride intravitreal injection increased the levels of phosphorylated Ser21/9 in GSK-3α/β and decreased TUNEL-positive cells in the whole-mounted retinas.

CONCLUSIONGSK-3 kinase is closely related to retinal neuron apoptosis, and the application of the GSK-3 inhibitor lithium chloride can reduce retinal neuron apoptosis in early diabetic retinopathy.

Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; Cell Survival ; physiology ; Diabetic Retinopathy ; enzymology ; genetics ; metabolism ; Flow Cytometry ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Male ; Neurons ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retina ; cytology ; enzymology

OBJECTIVETo optimize the hydrolysis process of linarin by response surface methodology, and to use the model of aldose reductase to study the acacetin's activity of aldose reductase inhibitory.

METHODThe model of acacetin enzyme in vitro was established by the determination of fluorescence absorption of NADPH, the inhibition rate of acacetin aldose reductase was calculated, and then the IC50 of hydrolysis was obtained. The hydrolysis process of linarin hydrolysis condition was optimized by using response surface method.

RESULTThe results indicated that the IC50 of acacetin (2.74 mg x L(-1)) was less than the IC50 of linarin (3.53 mg x L(-1)). Hydrolyzation time of 7.4 h, sulphuric acid concentration of 0.54 mol x L(-1) and the ratio of material to liquid of 3 : 1 were the optimum conditions.

CONCLUSIONHydrolyzate acacetin has preferable inhibitory activity of aldose reductase. The optimized hydrolysis condition of linarin is convenient to use with good predictability.

Aldehyde Reductase ; antagonists & inhibitors ; metabolism ; Animals ; Diabetic Retinopathy ; enzymology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Enzyme Inhibitors ; chemistry ; pharmacology ; Flavones ; chemistry ; Glycosides ; chemistry ; pharmacology ; Glycosylation ; Humans ; Hydrolysis ; Male ; Rats ; Rats, Wistar ; Retina ; enzymology

OBJECTIVETo investigate the role of Caspase-3 in retinal damage caused by light exposure in rats.

METHODSLight injury to retina was induced by persistent exposure to illumination (intensity: 30 000 +/- 50 lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1, 3, 7, and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis.

RESULTSThe examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, especially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pigment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day.

CONCLUSIONApoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

Animals ; Apoptosis ; radiation effects ; Caspase 3 ; genetics ; metabolism ; radiation effects ; Dose-Response Relationship, Radiation ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; radiation effects ; Light ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Retina ; enzymology ; pathology ; radiation effects ; ultrastructure