BACKGROUNDThe mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODSIn vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTSIn vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONSVEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

Animals ; Eye Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factors ; analysis ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; therapy ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology

We examined the localization of neurofascin (NF) in the sciatic nerve of rat. In the myelinated fibers, neurofascin localizes strongly in the nodal axolemma except the small central cleft and also expresses in the paranodes, and weakly in the Schmidt-Lanterman incisures. In the paranodes, NF localizes around the axolemma and it expresses in the apposing membrane of paranodal loops. Axoplasm, compact myelin and cytoplasm of Schwann cell do not express NF at all. In the Schmidt-Lanterman incisures, NF is expressed weakly along the Schwann cell membrane. We propose that neurofascin may be a plasmalemmal integral protein of Schwann cell in the paranode and plays some important roles for the maintenance of axo-glial junctions at the paranode. It may also have some roles for maintaining the structure of Schmidt-Lanterman incisure and have some relations with proteins localizing in the node.
Animals ; Cell Adhesion Molecules/*analysis/physiology ; Fluorescent Antibody Technique ; Microscopy, Immunoelectron ; Nerve Growth Factors/*analysis/physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve/*chemistry/ultrastructure

OBJECTIVETo observe the expression and distribution of adult rat axon guidance cues Netrin-1 and Slit2 at different time points after spinal cord injury and to investigate the guidance mechanism of regenerated axons.

METHODSTwenty adult Sprague Dawley (SD) rats were divided randomly into five groups with 4 in each. Four groups of them were used to make Allen's spinal cord punch models and we took materials randomly from one of them on the 2nd, 4th, 7th and 14th day respectively after operation. The left one group was taken as the control group. Immunofluorescence laser confocal scan was used to examine the co-expression and localization of Netrin-1 and Slit2 proteins in the injured site of the spinal cord.

RESULTSWithin two weeks after SCI, the expression of Netrin-1 and Slit2 proteins increased temporarily and there was co-expression of them on the neuron plasma membrane.

CONCLUSIONSSynchronous high expression and co-expression of axon attractant Netrin-1 and repellent Slit2 are found in the adult rat injured spinal cord in the damaged local and vicinity parts, and probably, they act as the key regulators of axon guidance regeneration.

Animals ; Female ; Intercellular Signaling Peptides and Proteins ; analysis ; Male ; Microscopy, Confocal ; Nerve Growth Factors ; analysis ; Nerve Regeneration ; physiology ; Nerve Tissue Proteins ; analysis ; Netrin-1 ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; Tumor Suppressor Proteins ; analysis

OBJECTIVETo detect the levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in aqueous humor of patients with active choroidal neovascularization (CNV).

METHODSAqueous humor samples were obtained from 32 patients with active CNV. The concentrations of VEGF and PEDF in aqueous humor were measured by enzyme linked immunosorbent assay (ELISA) for quantitative analysis. VEGF and PEDF in 10 samples of aqueous humor from patients with cataract were also detected by the same methods as control.

RESULTThe mean VEGF and PEDF concentrations in aqueous humor of active CNV patients were higher than those in the control group (P=0.000).

CONCLUSIONThe patients with active CNV exhibit significantly higher VEGF and PEDF levels than those in control, indicating that VEGF along with PEDF may modulate the formation of CNV.

Adult ; Aged ; Aged, 80 and over ; Aqueous Humor ; chemistry ; Choroidal Neovascularization ; metabolism ; Eye Proteins ; analysis ; Female ; Humans ; Male ; Middle Aged ; Nerve Growth Factors ; analysis ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis

OBJECTIVETo investigate the expression of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats with intrauterine growth retardation (IUGR) and its significance.

METHODSThe IUGR animal model was established by feeding rats low-protein diets during their pregnancy. Newborn rats were divided into catch-up growth, non-catch-up growth and control groups. Protein and mRNA levels of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats were determined by RT-PCR and Western blot, respectively.

RESULTSNesfatin-1/NUCB2 mRNA and protein were expressed in the gastric mucosa of rats immediately after birth, and their expression increased in an age-dependent manner in all three groups. Furthermore, the level of nesfatin-1/NUCB2 in the catch-up growth group was higher than that in the control group before weaning, whereas there was no significant difference in nesfatin-1/NUCB2 expression between the two groups after weaning. The level of nesfatin-1/NUCB2 in the non-catch-up growth group was lower than that in the catch-up growth group during the whole observation period. The level of ghrelin in the catch-up growth group was higher than that in the control group starting from day 12 after birth, whereas there was no significant difference in ghrelin expression between the two groups after weaning. The level of ghrelin in the non-catch-up growth group was lower compared with those in the catch-up growth and control groups from days 12 to 28 after birth.

CONCLUSIONSNesfatin-1 and ghrelin are co-expressed in the gastric mucosa of rats with IUGR after birth and interact with each other to produce long-term nutritional regulation.

Age Factors ; Animals ; Calcium-Binding Proteins ; analysis ; genetics ; DNA-Binding Proteins ; analysis ; genetics ; Female ; Fetal Growth Retardation ; metabolism ; Gastric Mucosa ; chemistry ; Ghrelin ; analysis ; genetics ; Male ; Nerve Tissue Proteins ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

Trisomy 21 (Down syndrome) is the most common congenital anomaly, and it occurs in one out of 700-1000 births. Current techniques such as amniocentesis and chorionic villi sampling (CVS) require lengthy laboratory culture procedures and high costs. This study was undertaken to establish a rapid prenatal diagnosis of trisomy 21 using real-time quantitative polymerase chain reaction (PCR) of fetal DNA from amniotic fluid. Real-time quantitative PCR was performed with DNA templates obtained from 14 normal blood samples, 10 normal amniotic fluid samples, 14 Down syndrome blood samples, and 7 Down syndrome amniotic fluid samples. Primers for D21S167 and S100B of chromosome 21 were used. Primers that direct the amplification of the 165-bp fragment of the insulin-like growth factor (IGF) -1 gene on chromosome 12 using a PCR primer were included to generate an internal standard for quantitation. The relative levels of D21S167 and S100B were 2.6 and 2.4 times higher in the blood of Down syndrome patients than those in the control group. The differences between these two groups were statistically significant (p-values were 0.0012 and 0.0016, respectively). The relative levels of D21S167 and S100B were 2.1 and 2.7 times higher in the amniotic fluid of Down syndrome fetuses than those in the control group. The difference between these two groups was statistically significant (p-values were 0.0379 and 0.0379, respectively). Prenatal diagnosis of trisomy 21 by real-time quantitative PCR using STR (small tandem repeats) amplification of D21S167 and S100B is a useful, accurate and rapid diagnostic method. Furthermore, it may also be useful for prenatal diagnosis with fetal DNA from maternal blood, and for preimplantation genetic diagnosis and prenatal counseling.
Amniotic Fluid/*chemistry ; Biological Markers/analysis ; Chromosomes, Human, Pair 21/*genetics ; Computer Systems ; Down Syndrome/*diagnosis ; Female ; Humans ; Nerve Growth Factors/*analysis ; *Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis/*methods ; S100 Proteins/*analysis ; *Tandem Repeat Sequences ; Time Factors

OBJECTIVE: To investigate the dynamics of the induction of S100beta in different parts of rat brain following the diffuse brain injury. METHODS: Immunohistochemistry and auto-image analysis were to determine the expression of astroglial S100beta after diffuse brain injury in rats. Forty rats were distributed into groups according to injury time of 30min, and2,4,12,24h, and 3,6 d after diffuse brain injury, and normal rats as control. RESULTS: The number of S100beta positive cells in the four areas increased significantly followed by a decrease, and then a further increase. The expression of S100beta could be detected increasing in 2h, and increased significantly in 4h, and it reached apex 12h after DBI, and decreased gradually to the level less than normal 3d, and returned to normal 7d following injury. In the postmortem injury groups, there were no significant changes in anti-S100beta immunoreactivities in four areas of brain compared to the control group. CONCLUSION The present study showed the time-dependent expression of S100beta is obvious following diffuse brain injury, and suggested S100beta be suitable as a marker for brain injury age determination.
Animals ; Brain/pathology* ; Brain Edema/pathology* ; Brain Injuries/pathology* ; Female ; Immunohistochemistry ; Male ; Nerve Growth Factors/analysis* ; Neuroglia/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; S100 Calcium Binding Protein beta Subunit ; S100 Proteins/analysis* ; Staining and Labeling ; Time Factors

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.
Adaptor Proteins, Signal Transducing ; analysis ; Adult ; Aggrecans ; analysis ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; CD146 Antigen ; analysis ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Collagen Type II ; analysis ; Core Binding Factor Alpha 1 Subunit ; analysis ; Flow Cytometry ; methods ; Homeodomain Proteins ; analysis ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; physiology ; Multipotent Stem Cells ; cytology ; physiology ; Nerve Tissue Proteins ; analysis ; Osteogenesis ; physiology ; Periodontal Ligament ; cytology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis ; SOX9 Transcription Factor ; analysis ; Time Factors ; Transcription Factors ; analysis

OBJECTIVETo investigate an effective treatment of peripheral nerve injuries by means of gene transference.

METHODS48 adult Wister rats were divided evenly into 3 groups. A 10 mm sciatic nerve gap was created and bridged with a silicone chamber. The silicone chamber was filled with glial cell-line derived neurotrophic factor(GDNF) gene modified Schwann cells(SCs) (group 1), the normal SCs(group 2) and nothing(the control). At 4, 8, 12, and 16 weeks after the operation, the general and histological observations, the electromyographic and immunohistochemical examinations were performed to the regenerated nerves.

RESULTSThe GDNF-SCs group was significantly better than the SCs and the control groups in nerve conduction velocity, the number and density of reinnervation, the area of regenerated nerve and the thickness of myelin sheath of the regenerated nerves.

CONCLUSIONGDNF gene modified SCs secrete higher levels of neurotrophic factors for a prolonged time, which are more effective in peripheral nerve repair than the normal SCs.

Animals ; Female ; Glial Cell Line-Derived Neurotrophic Factor ; Immunohistochemistry ; Male ; Nerve Growth Factors ; analysis ; genetics ; physiology ; Nerve Regeneration ; physiology ; Peripheral Nerves ; chemistry ; physiopathology ; Peripheral Nervous System Diseases ; surgery ; Rats ; Rats, Wistar ; Schwann Cells ; metabolism ; transplantation

OBJECTIVETo investigate the expression and pattern of vascular endothelial growth factor (VEGF) and its fetal liver kinase-1 (Flk-1) receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats.

METHODSForty-five adult male Wistar rats were divided randomly into a control group (n=5) and an experimental group (n=40). The bilateral sciatic nerves of the rats in the experimental group underwent neurotomy and the L4-L6 spinal cord and the corresponding dorsal root ganglia were harvested respectively at 8 hours, and 1, 3, 5, 7, 10, 14 and 21 days (8 subgroups with 5 rats each) after operation. The rats in the control group only underwent an exposure of sciatic nerve without neurotomy. Immunohistochemistry and image analysis were used to study the expression of VEGF and its Flk-1 receptor.

RESULTSBoth VEGF and Flk-1 receptor expressed in the normal rat spinal cord and dorsal root ganglia. In response to neurotomy, their expression reached a higher level and persisted for a short time then declined to the normal level rapidly. Besides, positive staining of Flk-1 was observed in both glial cells and nerve fibers, which located in the white matter of the spinal cord.

CONCLUSIONSVEGF can promote the regeneration of peripheral nerves from the angle of central neurons, which establishes the experimental and theoretical foundation for VEGF treating peripheral nerve injuries.

Analysis of Variance ; Animals ; Ganglia, Spinal ; metabolism ; Immunoenzyme Techniques ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; metabolism ; surgery ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; Vascular Endothelial Growth Factors ; biosynthesis