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

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

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 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

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 the effect of liposome-mediated glial cell line-derived neurotrophic factor (GDNF) gene transfer in vivo on spinal cord motoneurons after spinal cord injury (SCI) in adult rats.

METHODSSixty male Sprague-Dawley rats were divided equally into two groups: GDNF group and control group. The SCI model was established according to the method of Nystrom, and then the DC-Chol liposomes and recombinant plasmid pEGFP-GDNF cDNA complexes were injected into the injured spinal cord. The expression of GDNF cDNA 1 week after injection was detected by RT-PCR and fluorescence microscope. We observed the remaining motoneurons in the anterior horn and the changes of cholinesterase (CHE) and acid phosphatase (ACP) activity using Nissl and enzyme histochemistry staining. The locomotion function of hind limbs of rats was evaluated using inclined plane test and BBB locomotor scale.

RESULTSRT-PCR and fluorescence observation confirmed the presence of expression of GDNF cDNA 1 week and 4 weeks after injection. At 1, 2, 4 weeks after SCI, the number of motoneurons in the anterior horn in GDNF group (20.4+/-3.2, 21.7+/-3.6, 22.5+/-3.4) was more than that in control group (16.8+/-2.8, 17.3+/-2.7, 18.2+/-3.2, P<0.05). At 1, 2 weeks after SCI, the mean gray of the CHE-stained spinal motoneurons in GDNF group (74.2+/-25.8, 98.7+/-31.6) was less than that in control group (98.5+/-32.2, 134.6+/-45.2, P<0.01), and the mean gray of ACP in GDNF group (84.5+/-32.6, 79.5+/-28.4) was more than that in control group (61.2+/-24.9, 52.6+/-19.9, P<0.01). The locomotion functional scales in GDNF group were higher than that in control group within 1 to 4 weeks after SCI (P<0.05).

CONCLUSIONSGDNF gene transfer in vivo can protect motoneurons from death and degeneration induced by incomplete spinal cord injury as well as enhance locomotion functional restoration of hind limbs. These results suggest that liposome-mediated delivery of GDNF cDNA might be a practical method for treating traumatic spinal cord injury.

Animals ; Disease Models, Animal ; Gene Transfer Techniques ; Glial Cell Line-Derived Neurotrophic Factor ; Injections, Intralesional ; Liposomes ; Locomotion ; physiology ; Male ; Motor Neurons ; drug effects ; Nerve Growth Factors ; pharmacology ; Nerve Regeneration ; physiology ; Neuroprotective Agents ; pharmacology ; Primary Prevention ; methods ; Probability ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Recovery of Function ; Reference Values ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord Injuries ; pathology ; prevention & control ; therapy

OBJECTIVETo study the effects of ganglioside 1 (GM1) and nerve growth factor (NGF) on neural stem cells (NSCs) proliferation in vitro.

METHODSNSCs were isolated and cultured in vitro. NSCs were cultured in the medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) or without the two agents. Different concentrations of GM1 and NGF were added into the two different medium. MTT and cell ball counting methods were used to ascertain the proliferation of NSCs. Immunohistochemical technology was used to observe the effect of GM1 and NGF on the proliferation of NSCs.

RESULTSHigh concentrations of GM1 (100 ng/L and 200 ng/L) promoted significantly the proliferation of NSCs in the medium containing EGF and bFGF (p<0.05). In the differentiation medium containing serum but no EGF and bFGF, NSCs proliferation increased with increasing concentration of GM1; the proportion of neurons and gliacytes increased with increasing concentration of NGF.

CONCLUSIONSHigh concentration of GM1 can promote NSCs proliferation and NGF can promote NSCs differentiation.

Animals ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; G(M1) Ganglioside ; pharmacology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Growth Factors ; pharmacology ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; drug effects ; physiology