Background The vitreoretinal traction plays a critical role in the formation of macular hole and cystoid macular edema.Enzymatic vitreolysis has potential in relieving vitreoretinal traction as a simple and less invasive method in comparison with pars plane vitrectomy.ObjectiveThis study is to investigate the effects of plasmin mutant with kringle domains deficiency(Plm△K)on vitreoretinal interface in new Zealand white rabbits.Methods Plm△K was prepared through activating plasminogen mutant with Kringle domains deficiency (Plg△K) by tissue plasminogen activator (tPA).100μL of Plm△K at the dose of 0.5,1.0 and 1.5μmol/min was injected respectively into the vitreous of 48 New Zealand white rabbits and 16 eyes for each dose.B-scan and optical coherence tomography (OCT) were performed to detect the structure variety at the vitreoretinal interface in 1 day and 7 days after injection.The gross anatomy analysis with triamcinolone acetonide fine particle suspension,as well as histopathological examinations by scanning electron microscopy,was performed in the different time points mentioned above.Results Two peptide chains were determined with the relative molecular weight about 26000 and 5000 by the gel imaging analysis of reduced SDS-PAGE.Separation of the posterior vitreous cortex from retina was found after intravitreous injection under the B-scan and OCT.The ultrastructure change of vitreoretinal interface as well as the examination of fine particle suspension by triamcinolone acetonide demonstrated the same outcome.The degree of remnants of vitreous cortex showed the negative correlation with the dosage of Plm△K (r=-0.9516,P=0.048).No significant correlation was found between the degree of remnants of vitreous cortex and the action time(r=-0.720,P=0.470).There was no obvious morphological difference in outer layer of retina between control eyes and Plm△K-treated eyes.No drug-related adverse event was found after intravitreous injection of Plm△K.Conclusion Intravitreous injection of Plm△K alone can induce complete separation of vitreous from retina.This procedure is safe and effective.

Objective To study the efficacy and safety of different doses of caffeine citrate and aminophylline treatment for apnea of prematurity. Methods Preterm infants who met the inclusive criteria were admitted to NICU of JingZhou Central Hospital from October 1st, 2013 to October 1st, 2015. They were randomly assigned to three groups. Infants assigned to high dose caffeine group were received a loading dose of 40 mg / kg daily, followed by the maintaining dose of 20 mg / kg daily. Neonates in low dose caffeine group were administered with the loading dose of 20 mg / kg daily, followed by maintaining dose of 10 mg / kg daily. Infants in the aminophylline group received a loading dose of 5 mg / kg, then with maintaining dose of 2 mg / kg every 12 hours. Caffeine citrate or aminophylline therapy were continued until the infants were free from apnea for a period of 7 days or when the gestational age of 34 weeks were reached. Extubation failure rate, frequency of apnea, duration of apnea, mechanical ventilation, as well as oxygen therapy, length of hospital stay, mortality, and the adverse effects were compared among three groups. Results 90 infants were enrolled for study, with 30 in each group. Extubation failure rate, frequency of apnea, apnea duraion and oxygen therapy duration of infants in high dose caffeine groups were all significantly lower than those of infants in low dose caffeine group and aminophylline group (P < 0. 05). The incidence of tachycardia was significantly higher in the high dose caffeine group compared to the other two groups (P < 0. 05). Whereas all these factors between low dose caffeine group and aminophylline group were of no statistical difference (P > 0. 05). Duration of mechanical ventilation and CPAP, length of hospital stay, incidence of complications (BPD, ROP, IVH, PVL, NEC ), mortality were of no significant difference among three groups ( P > 0. 05 ) . Conclusions High dose caffeine therapy for apnea of prematurity is more effective in decreasing incidence of extubation failure and apnea, as well as decreasing duration of apnea and oxygen therapy. Tachycardia is the only adverse effect of high dose caffeine therapy discovered by this study.

AIM: To evaluate the effect of carbon monoxide(CO) on the permeability of brain blood barrier(BBB) in cerebral ischemic rats. METHODS: SD rats were divided into three groups. Saline, hemin or ZnPP were injected intraperitoneally 12 h before middle cerebral artery occlusion (MCAO), respectively. The concentration of blood CO and the permeability of BBB at 24 h after MCAO were measured. RESULTS: The CO concentration in blood in hemin group was higher than that in saline group( P 0.05). CONCLUSION: CO reduced the permeability of BBB as a messenger gas molecular when its intrinsic concentration was elevated.

Background Retinal vein occlusion is a common retinal vascular diseases.Thromblysis and anticoagulation therapies are main approaches.However,systemic thrombolysis is relatively inefficient,and it often enhances the risk of hemorrhage.Objective This study was to investigate the therapeutic effects of PLM-ΔK,a kringle deficiency mutant of plasmin,on photochemically induced branch retinal vein occlusion (BRVO) after intravitreal injection.Methods BRVO models were established by the combination of caudal vein injection of Rose Bengal with argon laser radiation of periphery area of retinal veins in SD rats.Forty model rats were randomized into balance salt solution (BSS) group and 0.01 U,0.02 U,0.03 U PLM-ΔK group,and 10 μl corresponding drug was intravtreally injected 12 hours after modeling.Ophthalmoscopy and fundus fluorescein angiography (FFA) were performed to observe the change of retinal veins.The animals were sacrificed 3 days after intravitreal injection,and hematoxylin and eosin staining was used for the histopathological and ultrastructural examination of retinas.The retina of the rats was isolated for the stretched preparation of retina.The expressions of fibronectin (FN) and laminin (LN) in eyeball wall were assayed by immunofluorescence technology.The use and care of the animals complied with Statement of the Association for Research in Vision and Ophthalmology.Results The revascularization of over 2 retinal veins was found in 0,3,6 and 8 rats in the BBS group and 0.01 U,0.02 U,0.03 U PLM-ΔK group 3 days after intravitreal injection,respectively,showing a significant difference among the groups (x2=9.635,P =0.022),and the rat number with revascularization in 0.01 U PLM-ΔK group was not significantly different from that in BSS group (Z=-1.558,P =0.119),but the difference between 0.03 U PLM-ΔK group and 0.01 U PLM-ΔK group was significant (Z=-2.762,P=0.006).In the third day after intravitreal injection,retinal vein thrombus were found in the BSS group under the light microscope,and angiogenesis was seen on the retinal flatmount nuclear.In the 0.03 U PLM-ΔK group,posterior vitreal detachment was exhibited under the light microcope,and no retinal new vessel and cell damage were seen.FN was strongly expressed in the inner limiting membrane (ILM) layer,photocyte layer,outer limiting membrane (OLM) layer,choroid and scleral layer,and LN was expressed mainly in the ILM,OLM and scleral layer in the BSS group.However,the expression intensities of FN and LN were obviously weakened in the 0.03 U PLM-ΔK group.Conclusions Intravitreal injection of PLM-ΔK can enhance the reperfusion of occluded branch retinal vein and serve as a potential therapeutic drug for BRVO.Also it can permeate into choroid after intravitreal injection to degradate FN and LN.

OBJECTIVESHeparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro.

METHODScDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth.

RESULTSIn the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells.

CONCLUSIONRecombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.

Animals ; Base Sequence ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Cytokines ; biosynthesis ; genetics ; pharmacology ; DNA, Complementary ; chemistry ; Genetic Vectors ; Humans ; Molecular Sequence Data ; Neurites ; drug effects ; physiology ; PC12 Cells ; Pichia ; genetics ; Rats ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction