Animals ; Ciliary Neurotrophic Factor ; genetics ; metabolism ; Microwaves ; RNA, Messenger ; genetics ; Rabbits ; Retina ; metabolism ; radiation effects

OBJECTIVETo investigate the changes of cholinergic neurotrophic factors (CNTF) protein at different time points and the distribution of CNTF in rabbit retina after exposure to high power microwave (HPM), in order to determine the changes rule of CNTF protein.

METHODSThe rabbits were irradiated by HPM (peak power 90 W/cm(2)) for 15 min respectively, and then killed at 0, 3, 6, 12, 24 and 72 h after irradiation. The changes of CNTF protein were investigated by immunohistochemistry and semi-quantity analysis.

RESULTSCNTF protein was distributed in full retinal layers, special in the cell membrane and cytoplasm. HPM irradiation could immediately down-regulated CNTF protein expression at 0 h, up-regulated and arrived at peak level at 6 h (P<0.05 vs 0 h group), and then kept control level.

CONCLUSIONHPM may cause acute retinal injure and change the expression of CNTF protein in rabbit retina. These effects show the time-dependent feature. These results suggest that CNTF activation plays a central role in the retinal injures induced by HPM, and supplies a therapy method by using foreign-aid CNTF to remedy the retinal injure induced by HPM.

Animals ; Ciliary Neurotrophic Factor ; metabolism ; Female ; Male ; Microwaves ; adverse effects ; Rabbits ; Retina ; metabolism ; radiation effects

Neural stem/progenitor cells (NSCs) can spontaneously differentiate into neurons and glial cells in the absence of mitogen fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) in medium and the spontaneous differentiation of NSCs is mediated partially by endogenous ciliary neurotrophic factor (CNTF). This study examined the relationship of FGF-2 and CNTF in the spontaneous differentiation of adult hippocampal progenitor cells (AHPs). AHPs were cultured in the medium containing different concentration of FGF-2 (1-100 ng/mL). Western blotting and immunofluorescence staining were applied to detect the expression of the astrocytic marker GFAP, the neuronal marker Tuj1, the oligodendrocytic marker CNPase and, Nestin, the marker of AHPs. The expression of endogenous CNTF in AHPs at early (passage 4) and late stage (passage 22) was also measured by Western blotting. The results showed that FGF-2 increased the expression of Nestin, dramatically inhibited the expression of GFAP and Tuj1 and slightly suppressed the expression of CNPase. FGF-2 down-regulated the expression of endogenous CNTF in AHPs at both early (passage 4) and late stage (passage 22). These results suggested that FGF-2 could inhibit the spontaneous differentiation of cultured AHPs by negatively regulating the expression of endogenous CNTF in AHPs.
Animals ; Cell Differentiation ; physiology ; Ciliary Neurotrophic Factor ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Hippocampus ; metabolism ; physiology ; Male ; Rats ; Rats, Wistar ; Stem Cells ; metabolism ; physiology

AIMTo investigate the effect of ciliary neurotrophic factor (CNTF) on the nuclear translocation of protein kinase C (PKC) following NMDA administration in the primary cultured hippocampal neurons.

METHODS(1) PKCGAMMA or PKCepsilon- immunocytochemistry staining method was used after treating neurons with NMDA or CNTF. (2) The gray of the nucleus of the PKC-positive neurons were measured under the image pattern analysis system.

RESULTS(1) After NMDA administration of different concentration and time, Nucleus appear PKCgamma and PKCepsilon activities, especially the 100 micromol/L NMDA 30 min group. (2) The gray of nucleus in CNTF + 500 micromol/L NMDA group is similar to control group.

CONCLUSIONNMDA can induce nuclear translocation of PKC in the primary cultured hippocampal neurons, and CNTF can inhibit the translocation. It suggests that the inhibition of PKC translocation induced by NMDA is one of the important reasons for the neuro-protective effects of CNTF.

Animals ; Cells, Cultured ; Ciliary Neurotrophic Factor ; pharmacology ; Hippocampus ; cytology ; N-Methylaspartate ; pharmacology ; Neurons ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Protein Transport ; Rats ; Rats, Sprague-Dawley

BACKGROUNDGlaucoma is mainly characterized by the loss of retinal ganglion cells. Ciliary neurotrophic factor (CNTF) is believed to stimulate the regeneration of axons of retinal ganglion cells. The objective of our study was to detect the expression of CNTF in the retina of a rat glaucoma model with increased intraocular pressure (IOP).

METHODSThe rat glaucoma model was set up by electrocoagulating at least three episcleral and limbal veins. The location and the expression level of CNTF were detected at 1, 3, 7, 14, and 28 days post-surgery by immunohistochemistry, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), and Western blot analysis.

RESULTSThe rat glaucoma model with chronic, moderately elevated IOP was successfully produced. A minimum expression of CNTF was found in the ganglion cell layer of the retinas of the control group, and temporally increased expression and intensity of CNTF were found in the experimental retinas.

CONCLUSIONThe expression of endogenous CNTF in the rat retina was found altered after the induction of ocular hypertension.

Animals ; Ciliary Neurotrophic Factor ; analysis ; genetics ; Densitometry ; Immunoblotting ; Immunohistochemistry ; Male ; Ocular Hypertension ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Retina ; metabolism ; ultrastructure ; Reverse Transcriptase Polymerase Chain Reaction

To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Animals ; Ciliary Neurotrophic Factor ; genetics ; Humans ; Mice ; Mutant Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; genetics

AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Mutation ; Peptide Hydrolases ; chemistry ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

The distribution of the nerve growth factor (NGF), the glial fibrillary acidic protein (GFAP) and the ciliary neurotrohic factor (CNTF) was performed in coronal sections of the mesencephalon, rhombencephalon and spinal cord in the developing Mongolian gerbils. Generally, NGF specifically recognizes neurons with the NGF receptor, whereas GFAP does the glia, and CNTF does the motor neurons. The receptor expression was examined separately in gerbils between embryonic days 15 (E15) and postnatal weeks 3 (PNW 3). The NGF-IR was first observed in the spinal cord at E21, which might be related to the maturation. The GFAP reactivity was peaked at the postnatal days 2 (PND2), while the highest CNTF-reaction was expressed at PNW 2. The GFAP stains were observed in the aqueduct and the spinal cord, which appeared to project laterally at E19. The CNTF was observed only after the birth and found in both the neurons and neuroglia of the substantia nigra, mesencephalon, cerebellum and the spinal cord from PND1 to PNW3. These results suggest that NGF, GFAP and CNTF are important for the development of the neurons and the neuroglia in the central nervous system at the late prenatal and postnatal stages.
Animals ; Brain Stem/enzymology/*metabolism ; Ciliary Neurotrophic Factor/*metabolism ; Embryonic and Fetal Development/physiology ; Female ; Gerbillinae/*embryology ; Glial Fibrillary Acidic Protein/*metabolism ; Immunohistochemistry/veterinary ; Mesencephalon/embryology/metabolism ; Nerve Growth Factor/*metabolism ; Pregnancy ; Rhombencephalon/embryology/metabolism ; Spinal Cord/embryology/*metabolism

OBJECTIVEThe aim of this study was to observe the expression of ciliary neurotrophic factors (CNTFs) in the facial motor neurons in rats during facial nerve regeneration.

METHODSThe expression amount of CNTFs in eight groups was determined with immunohistochemical staining and image analysis.

RESULTSThe expression of CNTFs increased during the process of nerve regeneration, and reached the maximum one-week and one-month after nerve injury respectively. After one month, the intensity of CNTFs reduced gradually. The expression of recombinant human bone morphogenetic protein-2 (rhBMP-2) did not show distinct difference comparing with the control, but beta transforming growth factors (TGF-beta) benefited the expression of CNTFs during nerve regeneration.

CONCLUSIONThe endogenous CNTFs promotes the axon outgrow during regeneration, and TGF-beta promotes the expression of CNTFs to rescue motoneuron during facial nerve regeneration.

Animals ; Bone Morphogenetic Proteins ; metabolism ; Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; Facial Nerve ; metabolism ; physiopathology ; Facial Nerve Injuries ; physiopathology ; Male ; Motor Neurons ; metabolism ; Nerve Regeneration ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration.

METHODSD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later.

RESULTMost of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA.

CONCLUSIONTraditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

Animals ; Arecaceae ; chemistry ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cells, Cultured ; Ciliary Neurotrophic Factor ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; GAP-43 Protein ; genetics ; metabolism ; Gene Expression ; drug effects ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Male ; Nerve Growth Factor ; genetics ; metabolism ; Nerve Regeneration ; drug effects ; Neurofilament Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, trkA ; genetics ; metabolism ; Schwann Cells ; drug effects ; physiology ; Serum ; chemistry