OBJECTIVE: To investigate the effect of pretreatment with neurotrophin-3 (NT-3) on intrathecal ropivacaine in rats. METHODS: A total of 144 male Sprague Dawley rats weighing 280-320 g were successfully implanted with microspinal cather following the improved methods of Yaksh. The rats were randomly divided into 4 groups and given saline (Group NS, n=36), 0.5% ropivacaine (Group M, n=36), 1% ropivacaine (Group R, n=36), and ropivacaine+NT-3 (Group T, n=36). The rats received 0.12 mL/ kg body weight of ropivacaine at 0.5% or 1%, or normal saline only, via an implanted intrathecal catheter at 90-min interval for 12 h in Group NS, M, R and T. In the meantime the rats also received NT-3 0.1 mg/kg in group T. On days 1, 3, 5, 7, 14 and 28, we assessed the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), behavioural change and histopathological damage score changed for possible neuronal injury within the spinal cord. RESULTS: Compared with Group NS and Group M, the PWMT and PWTL were significantly higher on 1, 3, 5 d and the histopathological damage score was significantly higher on 1, 3, 5, 7, 14 d in Group R (P<0.05). Compared with Group T, the PWMT and PWTL in Group R were significantly higher on 1, 3, 5 d and histopathological damage score was significantly higher on 5, 7, 14 d (P<0.05). CONCLUSION NT-3 pretreatment in mice has obvious protective effect against repeated intrathecal injection of 1% ropivacaine in the spinal nerve.
Amides ; adverse effects ; Animals ; Injections, Spinal ; Male ; Neurotrophin 3 ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ropivacaine ; Spinal Cord ; drug effects

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

OBJECTIVETo explore the method for obtaining olfactory ensheathing cells from human fetal olfactory mucosa by cell culture for selective adhesion in the presence of neurotrophin-3 (NT3) and low-concentration serum.

METHODSThe olfactory ensheathing cells were cultured alternatively in DMEM/F12 culture medium containing 10% fetal bovine serum (FBS) and the medium containing NT3 and 2.5% FBS every 72 h. The cells were observed for morphological changes and identified using immunocytochemistry with P75NTR and GFAP, and the cell purity was estimated.

RESULTSThe olfactory ensheathing cells from human fetal olfactory mucosa were positive for P75(NTR) and GFAP, and in in vitro culture, the cells exhibited dipolar or tripolar appearance with long thin neurites. On the 9th day of cell culture, the purity of the olfactory ensheathing cells reached about 83%.

CONCLUSIONThe olfactory ensheathing cells can be obtained by in vitro culture for selective adhesion in the presence of NT3 and low-concentration serum.

Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cells, Cultured ; Culture Media ; Fetus ; Humans ; Neurotrophin 3 ; pharmacology ; Olfactory Bulb ; cytology ; Olfactory Mucosa ; cytology

OBJECTIVE: This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). METHODS: hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. RESULTS: Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. CONCLUSIONS Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.
Alkaline Phosphatase ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; Humans ; Mesenchymal Stem Cells ; Neurotrophin 3 ; pharmacology ; Osteocalcin ; metabolism ; Osteogenesis

OBJECTIVETo observe the protective effect of nitric oxide synthase inhibitor-N(G)-nitro-L-arginine methyl ester (L-NAME) with or without neurotrophin 3 (NT3) on hearing in acoustic trauma.

METHODSEighty pigmented male guinea pigs were randomly divided into two groups: sham-exposed group (n=20) and noise-exposed group. The latter was divided into three subgroups: saline group (n=20), L-NAME group (n=20) and L-NAME + NT3 group (n=20). Two days consecutively and 30 min before noise exposure (4 kHz octave band noise at 115 dB SPL for 5 h), subjects in L-NAME and L-NAME + NT3 groups received an intraperitoneal injection of 10 mg/kg; animals in saline group received the same dosage of physiological saline at the same time. Four days before noise exposure, NT3 in artificial perilymph was delivered to the right scala tympani via a mini-osmotic pump in noise + L-NAME + NT3 group. Auditory brainstem responses (ABR) were measured before and 10 days following noise exposure. The cochlear tissue was assayed for nitric oxide (NO) level 3 days after noise exposure. Protection was assessed physiologically by the change in ABR threshold shift, and histologically by outer hair cell (OHC) survival.

RESULTSThe hearing thresholds and the number of OHC were relatively stable in sham-exposed group. The obvious threshold shift and OHC loss were observed in the noise-exposed groups. The hearing thresholds, NO level of cochlear tissue and OHC loss in the noise + saline group were significantly higher than those in the noise + L-NAME group (P < 0.01) and noise + L-NAME + NT3 group (P < 0.01). NT3 provided an additive functional (P < 0.01), but not morphological protection with L-NAME (P = 0.095).

CONCLUSIONCompared to L-NAME alone, a combination of L-NAME and NT-3 can provide an additional protection against acoustic trauma in the guinea pig cochlear.

Animals ; Cochlea ; drug effects ; injuries ; Enzyme Inhibitors ; pharmacology ; Guinea Pigs ; Hair Cells, Auditory ; drug effects ; Hearing Loss, Noise-Induced ; prevention & control ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Neurotrophin 3 ; pharmacology ; Nitric Oxide Synthase ; antagonists & inhibitors

OBJECTIVETo investigate the effect of extract of Ginkgo Biloba(EGB) on nerve growth factor(NGF) and Neurotrophin-3(NT-3) expression of hippocampus neurons in streptozotocin-induced type I diabetic rats.

METHODSThirty male SD rats were divided into three groups (n = 10): the control group, diabetic group and EGB-treated group. Strepozotocin were injected intraperitoneally in the later two groups to induce diabetes. EGB-treated group was injected intraperitoneally with EGB, and the same volume of normal saline was injected to the other groups. Concentration of blood glucose and body weight and behaviour were dynamicly monitored. At the end of the 12th week, morphological changes of the hippocampus neurons were observed under microscopy by HE stain. The expression of NGF and NT-3 were assayed by Western blot and RT-PCR respectively.

RESULTSCompared with diabetic group, the behaviour and body weight (P < 0.05) and the concentration of blood glucose (P < 0.05) were significantly improved and the escape latency of Morris water maze test (P < 0.05) was significantly shortened, while the platform searching score was significantly increased (P < 0.01) in EGB treated group; The pathological changes of hippocampus neurons were significantly attenuate by EGB treated; The expression of NGF and NT-3 in hippocampus neurons were significantly increased which assayed by Western blotting and RT-PCR respectively (P < 0.05) in EGB treated group.

CONCLUSIONEGB may improve the learning and memory ability of diabetic rats the mechanism may be attributed to its improvement of the expression of NGF and NT-3 and reducing apoptosis in hippocampus neurons.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; psychology ; Ginkgo biloba ; Hippocampus ; cytology ; Male ; Maze Learning ; drug effects ; Nerve Growth Factor ; metabolism ; Neurons ; drug effects ; metabolism ; Neurotrophin 3 ; metabolism ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To transfect the recombinant plasmid enhancement type green fluorescent protein C2- neurotrophic factor-3 (pEGFPC2-NT3) into the spinal ganglion cells(SGCs) of guinea pigs' cochlea injured by the excitotoxicity of hydroxyapatite particle (HAT), to inject the recombinant plasmid pEGFPC2-NT3 into the guinea pigs' cochlea, and to observe the expression of pEGFPC2-NT3 and the protective effect of pEGFPC2-NT3 on SGCs of the cochlea in guinea pigs. METHODS: The recombinant plasmid pEGFPC2-NT3 with gene-green fluorescent protein was established. Kanic acid (KA) was injected into guinea pigs'cochleae and the excitotoxicity model was established. After a week the recombinant plasmid was transferred into SGCs of guinea pigs'cochlea treated with HAT. The following week the expression of NT-3 was examined by the immunohistochemical method, and the morphology of SGNs was observed under the electronic microscope after 4 weeks, in the mean time the changes of auditory brain-stem response (ABR) were examined. RESULTS: The excitotoxicity models were established successfully. NT-3 expression in the intracytoplasm of SGNs was observed by the immunohistochemical method 1 week after the injection, the morphologic damages of SGNs lessened under the electronic microscope after 4 weeks. ABR was partly restored, compared with ABR after the injury of the excitotoxicity. CONCLUSION On the 7th day, NT3 gene transferred by HAT through the scala tympani can lessen the excitotoxicity of SGCs after KA was injected into the guinea pigs cochlea.
Animals ; Cochlea ; drug effects ; Durapatite ; administration & dosage ; pharmacology ; Evoked Potentials, Auditory, Brain Stem ; Ganglia, Spinal ; drug effects ; metabolism ; Genetic Therapy ; Guinea Pigs ; Kainic Acid ; adverse effects ; Nanocomposites ; Neurotoxins ; adverse effects ; Neurotrophin 3 ; genetics ; Plasmids

PURPOSE: To investigate the effect of macrophage migration inhibitory factor (MIF) on corneal sensitivity after laser in situ keratomileusis (LASIK) surgery. METHODS: New Zealand white rabbits were used in this study. A hinged corneal flap (160-microm thick) was created with a microkeratome, and -3.0 diopter excimer laser ablation was performed. Expressions of MIF mRNA in the corneal epithelial cells and surrounding inflammatory cells were analyzed using reverse transcription polymerase chain reaction at 48 hours after LASIK. After LASIK surgery, the rabbits were topically given either 1) a balanced salt solution (BSS), 2) MIF (100 ng/mL) alone, or 3) a combination of nerve growth factor (NGF, 100 ug/mL), neurotrophine-3 (NT-3, 100 ng/mL), interleukin-6 (IL-6, 5 ng/mL), and leukemia inhibitory factor (LIF, 5 ng/mL) four times a day for three days. Preoperative and postoperative corneal sensitivity at two weeks and at 10 weeks were assessed using the Cochet-Bonnet esthesiometer. RESULTS: Expression of MIF mRNA was 2.5-fold upregulated in the corneal epithelium and 1.5-fold upregulated in the surrounding inflammatory cells as compared with the control eyes. Preoperative baseline corneal sensitivity was 40.56 +/- 2.36 mm. At two weeks after LASIK, corneal sensitivity was 9.17 +/- 5.57 mm in the BSS treated group, 21.92 +/- 2.44 mm in the MIF treated group, and 22.42 +/- 1.59 mm in the neuronal growth factors-treated group (MIF vs. BSS, p < 0.0001; neuronal growth factors vs. BSS, p < 0.0001; MIF vs. neuronal growth factors, p = 0.815). At 10 weeks after LASIK, corneal sensitivity was 15.00 +/- 9.65, 35.00 +/- 5.48, and 29.58 +/- 4.31 mm respectively (MIF vs. BSS, p = 0.0001; neuronal growth factors vs. BSS, p = 0.002; MIF vs. neuronal growth factors, p = 0.192). Treatment with MIF alone could achieve as much of an effect on recovery of corneal sensation as treatment with combination of NGF, NT-3, IL-6, and LIF. CONCLUSIONS: Topically administered MIF plays a significant role in the early recovery of corneal sensitivity after LASIK in the experimental animal model.
Animals ; Epithelium, Corneal/*drug effects/innervation/physiology ; Female ; Humans ; Interleukin-6/pharmacology ; Keratomileusis, Laser In Situ/*methods ; Leukemia Inhibitory Factor/pharmacology ; Macrophage Migration-Inhibitory Factors/genetics/*pharmacology ; Models, Animal ; Nerve Growth Factor/pharmacology ; Nerve Regeneration/*drug effects/physiology ; Neurotrophin 3/pharmacology ; RNA, Messenger/metabolism ; Rabbits ; Recovery of Function/*drug effects/physiology ; Sensation/*drug effects/physiology

BACKGROUNDPrevious work has shown that optic nerve and sciatic nerve conditional medium had neurotrophic activity on neurons. In order to find if the optic nerve conditioned media (CM) had a similar activity to make PC12 cells differentiate as sciatic nerve CM did, we explored the neurotrophic activity in optic nerve CM in the same in vitro system and compared the neurotrophin expression levels in optic and sciatic nerves under both conditions.

METHODSPC12 cells were used to examine the effects of neurotrophins secreted by the sciatic nerve and optic nerve. RT-PCR and real-time QPCR showed that the sciatic nerve and optic nerve produced a range of neurotrophins including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).

RESULTSThe effects of sciatic nerve and optic nerve CM on neurite outgrowth were tested against a range of neurotrophins, and they had different neuritogenic activities. Only NGF and sciatic nerve CM had obvious neuritogenic activities, although the concentration of NGF in the sciatic nerve CM was very low.

CONCLUSIONSOur experiment showed that sciatic nerve CM had a higher neurotrophic activity on PC12 cells than optic nerve CM. These results suggested that peripheral nervous system (PNS) and central nervous system (CNS) had different expression levels of neurotrophin, which may in part explain the lack of ability to regenerate the CNS.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; pharmacology ; Cell Differentiation ; drug effects ; Culture Media, Conditioned ; metabolism ; pharmacology ; Nerve Growth Factor ; genetics ; pharmacology ; Neurotrophin 3 ; genetics ; pharmacology ; Optic Nerve ; metabolism ; PC12 Cells ; cytology ; drug effects ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Sciatic Nerve ; metabolism